异源四倍体油菜<i>HAKs</i>家族核心基因的鉴定及其功能初步解析

陈俊帆; 宋海利; 周婷; 岳彩鹏; 冯英娜; 黄进勇; 华营鹏

doi:10.11674/zwyf.2023414

异源四倍体油菜HAKs家族核心基因的鉴定及其功能初步解析

陈俊帆^{1, 2,},
宋海利^{1, 2},
周婷^{1, 2},
岳彩鹏^{1, 2},
冯英娜^{1, 2},
黄进勇^{2, 3, 4, ,},
华营鹏^{1, 2, ,}

1.
郑州大学农学院，河南郑州 450001
2.
郑州市作物品质改良与养分高效利用重点实验室，河南郑州 450001
3.
郑州大学生命科学学院，河南郑州 450001
4.
河南省伏牛山生物资源与生态环境野外科学观测研究站，河南郑州 450001

基金项目: 国家自然科学基金项目 (31801923)。

详细信息

作者简介:
陈俊帆 E-mail: hyp19890413@163.com

通讯作者:
黄进勇 E-mail: jinyhuang@zzu.edu.cn

华营鹏 E-mail: yingpenghua@zzu.edu.cn

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- 文章访问数: 161
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出版历程
- 收稿日期: 2023-09-20
- 录用日期: 2023-11-15
- 网络出版日期: 2024-03-27
- 刊出日期: 2024-03-24

Identification of the core member of the HAKs family and primary analysis on their functions in allotetraploid rapeseed (Brassica napus L.)

CHEN Jun-fan^{1, 2,},
SONG Hai-li^{1, 2},
ZHOU Ting^{1, 2},
YUE Cai-peng^{1, 2},
FENG Ying-na^{1, 2},
HUANG Jin-yong^{2, 3, 4, ,},
HUA Ying-peng^{1, 2, ,}

1.
School of Agricultural Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
2.
Zhengzhou Key Laboratory of Quality Improvement and Efficient Nutrient Use for Main Economic Crops, Zhengzhou, Henan 450001, China
3.
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China
4.
Henan Funiu Mountain Biological and Ecological Environment Observatory, Zhengzhou, Henan 450001, China

摘要

摘要:
目的
HAKs家族基因编码高亲和力K⁺转运蛋白，在K⁺吸收和运输过程中发挥关键作用。鉴定甘蓝型油菜HAKs家族核心基因，研究不同养分胁迫下核心基因表达的响应，不仅可加深对HAKs功能的理解，也可为HAKs介导油菜营养胁迫抗性的遗传改良提供基因资源参考。
方法
对甘蓝型油菜HAKs基因家族进行全基因组鉴定和分析，其中包括系统发育关系、基因结构、保守基序、染色体定位、顺式作用元件。利用不同养分胁迫下的转录组数据对HAKs家族基因进行差异基因表达分析，并用Cytoscape构建共表达网络图。以低钾抗性品系“H280”和低钾敏感品系“L49”为材料，在低钾胁迫下进行水培试验，使用ICP-MS分析了“H280”和 “L49”根部K⁺含量，RT-qPCR以及亚细胞定位分析了低钾胁迫下BnaA7.HAK5的响应和功能。
结果
在甘蓝型油菜中共鉴定到40个HAKs家族成员。对其进行进化关系分析，发现它们的Ka/Ks值均小于1.0。顺式作用元件分析表明，MYB和激素响应顺式作用调控元件ABRE在HAKs家族基因的启动子区域高度富集。共线性分析表明在进化过程中，甘蓝型油菜中绝大多数的HAKs基因保存完整。差异表达基因分析发现，大部分HAKs家族基因的表达水平受低钾胁迫的显著诱导。共表达网络分析表明，BnaA7.HAK5响应油菜低钾胁迫。RT-qPCR结果显示，BnaA7.HAK5在抗低钾品系“H280”根中的表达量显著高于低钾敏感品系“L49”。亚细胞定位结果显示，BnaA7.HAK5定位在细胞质膜上。
结论
甘蓝型油菜共有40个BnaHAKs基因，BnaHAKs基因响应低钾或盐等养分胁迫。定位在细胞质膜上的BnaA7.HAK5在低钾条件下的表达量显著上调，且在“H280”和“L49”油菜品系根部的表达量差异显著，表明BnaA7.HAK5在油菜高亲和性钾离子的吸收和转运中发挥重要功能。
- 甘蓝型油菜 /
- 低钾胁迫 /
- HAKs家族基因 /
- 基因组鉴定 /
- 转录组学
Abstract:
Objectives
The HAKs (high-affinity K transporter) family genes encode high-affinity potassium (K⁺) transporters, and play key roles in the absorption and transport of K⁺. Identification of the core genes of the HAKs family in rapeseed, and the study of their expression responses when exposure to different nutrient stresses, will help fully understanding of the functions of HAKs, and also provide valuable genetic resource references for the genetic improvement of HAKs-mediated nutrient resistance in rapeseed.
Methods
The genome-wide identification and analysis of the rapeseed HAKs gene family were made from aspects of phylogenetic relationships, gene structure, conserved motifs, chromosomal localization, cis-acting elements. Differential expression of HAKs family genes were analyzed using transcriptomic data under different nutrient stresses, and the co-expression network was constructed by Cytoscape. Low-K-resistant line “H280” and low-K-sensitive line “L49” were subjected to K stress in a hydroponic experiment, the K⁺ contents in the roots of line “H280” and “L49” were analyzed using ICP-MS. The response and function of BnaA7.HAK5 under low-K⁺ stress were analyzed by RT-qPCR and subcellular localization.
Results
A total of 40 HAKs family members were identified through genome-wide identification. Evolutionary relationship analysis of the HAKs family genes showed that their Ka/Ks values were all less than 1.0. The MYB and the hormone response cis-acting regulatory element ABRE were highly enriched in the promoter region of the HAKs family genes. Collinear analysis showed that most of the HAKs genes in Brassica napus were intact during the evolutionary process. Differential expression gene analysis revealed that the expression levels of most HAKs family genes were significantly induced by low potassium stress. The co-expression network analysis showed that BnaA7.HAK5 responded to low potassium stress in oilseed rape. The RT-qPCR results showed that the expression level of BnaA7.HAK5 in the roots of line “H280” was significantly higher than that of line “L49”. The results of subcellular localization showed that BnaA7.HAK5 was localized on the plasma membrane of cells.
Conclusions
There are 40 BnaHAKs genes in rapeseed, and the BnaHAKs genes respond to nutrient stresses such as low potassium or salt. BnaA7.HAK5, localized in the plasma membrane, is significantly up-regulated under low-potassium conditions, and showed differential expressions in the roots between “H280”and “L49” oilseed rape lines. It could be concluded that BnaA7.HAK5 plays an important role in the high-affinity uptake and transport of potassium ions in rapeseed.
- Brassica napus /
- low-potassium stress /
- HAKs family genes /
- genome identification /
- transcriptomics

HTML全文

钾(K)是植物必需的营养元素之一，作为一种重要的无机渗透剂，在蛋白质合成、糖和脂类的代谢、韧皮部运输、渗透调节、光合作用和植物生长发育中发挥重要的作用^[1−7]。在K⁺缺乏状态下，植物往往表现出叶片干枯、倒伏和坏死的迹象，从而影响农作物的产量和品质^[8]。另外，K⁺还能够增强植物对生物和非生物胁迫的抵抗力，改善其生长状况^[9−10]。油菜(Brassica napus L., AnAnCnCn, 2n= 4x= 38)是重要的油料作物，需要大量的K⁺来维持其生长和产量， K⁺需求量为290～373 kg/hm^2[11−12]。然而，我国是一个钾盐资源极为缺乏的国家，尤其是在我国的南方地区，由于土壤风化淋溶强烈，导致土壤含钾量低^[13]。因此，提高油菜K⁺的有效吸收将有助于作物的可持续生产和高品质作物的培育。

HAK/KUP/KT (HAK)是植物中最大的K⁺转运蛋白家族，其主要作用是负责钾离子的吸收和转运^[14−15]。根据蛋白对K⁺亲和性的不同，HAKs家族被分为3种类型：第一类是高亲和性钾离子转运体，例如拟南芥的AtHAK5^[16]、大麦的HvHAK1^[17]、水稻的OsHAK1^[18]均与根系高亲和K⁺的吸收有关，另外拟南芥的AtKUP7^[19]、水稻的OsHAK11^[20]、番茄的LeHAK5^[21]在K⁺缺乏时被强烈诱导；第二类是低亲和性钾离子转运体，如AtKUP2^[22]、大麦的HvHAK2^[23]和海草的CnHAK1^[24]，其作用是在外界高钾时介导K⁺的吸收和转运；第三类是双亲和性钾转运体，以AtKUP1为代表，其作用是在低钾和高钾条件下对K⁺吸收，促使植物正常生长^[25−26]。HAKs家族不仅促进植物钾离子吸收，而且HAKs家族的各种成员在植物非生物胁迫中也发挥着至关重要的作用。例如，拟南芥AtKUP6和水稻OsHAK1能提高植物的耐旱性^[27−28]，拟南芥AtHAK5、AtHAK6、水稻OsHAK21和玉米ZmHAK4能提高植物的耐盐性^[29−31]。此外，HAKs家族还参与植物的生长和发育过程。例如，OsHAK16可控制水稻株高的生长^[32]，OsHAK1控制水稻种子的发芽率以及圆锥花序的发育^[33]；AtKUP2、AtKUP6和AtKUP8调节K⁺外流，从而负调控植株的生长^[34]。

HAKs家族成员定位在植物不同细胞器的膜上，如细胞质膜、液泡膜、类囊体膜等^[35]。随着大量基因组测序完成，可将该基因家族划分为5个亚族。亚族I成员以拟南芥AtHAK5为代表，促进植物根系在外部低浓度K⁺的环境中吸收钾^[36−37]。亚族II成员因其转运及生理功能的不同而具有功能多样性。对于单子叶植物来说，许多亚族II的成员常常会参与到低亲和性K⁺吸收过程中，而在双子叶植物中则具有不同的转运活性，例如：在拟南芥中，AtKUP6介导低亲和性K⁺吸收^[38]，AtKUP4/TRH1介导高亲和性K⁺吸收^[39]，AtKUP1介导兼性K⁺吸收^[40]。然而，关于亚族III、IV和V三者的研究还很少。

本研究对甘蓝型油菜中HAKs家族进行全基因组鉴定和分析，主要包括基因结构、染色体定位、共线性、顺式作用元件、系统进化等方面的生物信息学分析。随后，针对低钾胁迫下转录组数据，对HAKs家族成员的表达谱进行分析，并结合基因共表达网络分析筛选出HAKs家族成员中响应低钾胁迫的核心成员，为深入研究甘蓝型油菜HAKs家族的功能奠定基础，并为未来研究耐低钾油菜品种奠定理论基础。

1. 材料与方法

1.1 HAKs家族基因序列的检索及命名

本研究从拟南芥信息资源(TAIR10, https://www.arabidopsis.org/)下载了拟南芥(A. thaliana) HAKs家族的CDS、蛋白序列和ID号，从芸薹属数据库(BRAD) V3.0 (http://www.brassicadb.cn/)下载甘蓝型油菜B. napus(Brana_Dar_V5基因组)、甘蓝(B. oleracea)、白菜(B. rapa)的基因组和蛋白质序列^[41−42]。利用已知的13个AtHAKs蛋白序列与甘蓝型油菜蛋白序列进行本地BLASTP比对，设定E-value<1e^-10，筛选出同源性较高的序列^[43−44]。并使用Pfam (https://www.ebi.ac.uk/interpro/entry/pfam/#table)在线工具，进一步筛选，剔除没有保守结构域的蛋白序列，最终筛选得到甘蓝型油菜HAKs家族的所有成员^[45]。甘蓝和白菜中HAKs家族成员鉴定方法同上。

本研究中BnaHAKs家族基因的命名规则为：种名缩写+染色体编号(后接句号)+拟南芥同源基因名称。如果同一条染色体上有多个同源基因，则分别用a、b、c等表示。例如，BnaA7.HAK5代表油菜A7染色体上的拟南芥HAK5的同源基因^[46]。

1.2 HAKs家族基因的理化性质分析及染色体定位

ExPASY-ProtParam在线工具(https://web.expasy.org/protparam/)用于分析HAKs家族基因的基本理化性质^[47−48]，主要包括氨基酸数量、脂肪族氨基酸指数(AI)、等电点(pI)、亲水性总平均值(GRAVY)、脂肪族指数和不稳定性指数(II)，II值>40.0表示该蛋白质不稳定^[49]。为了预测HAKs家族基因的跨膜结构域，将HAKs家族基因的蛋白序列提交给在线网站TMHMM (http://www.cbs.dtu.dk/services/TMHMM/)。为了预测HAKs家族基因的亚细胞位置，将HAKs家族基因的氨基酸序列提交到在线软件WoLF PSORT (https://www.genscript.com/wolf-psort.html)和TargetP-2.0 (https://services.healthtech.dtu.dk/services/TargetP-2.0/)^[50]。

利用HAKs家族基因的完整核苷酸序列，通过BLASTn搜索并确定HAKs家族基因在基因组中的位置。并通过MapInspect v. 2010 (http://www.softsea.com/review/MapIn-spect.htmL)软件用于绘制这些基因的染色体图谱。

1.3 HAKs家族基因的共线性分析及选择压力分析

利用TBtools软件对甘蓝型油菜、甘蓝、白菜、拟南芥的HAKs家族基因进行共线性分析，并使用TBtools内置插件One Step MCScanx-Super Fast绘制共线性关系图^[51]。为了判断是否存在选择性压力作用于HAKs家族基因，本研究计算了非同义取代率(Ka)、同义取代率(Ks)和Ka/Ks值。首先，使用在线软件Clustal Omega (http://www.clusta-l.org/omega/)对HAKs家族基因的CDS进行成对比对^[52]，然后将这些结果提交给Ka/Ks计算器(https://sourceforge.net/pro-jects/kakscalculator2/)，用yn00方法计算Ka、Ks和Ka/Ks值^[53]。如果Ka/Ks>1.0，则认为存在正选择效应；如果Ka/Ks = 1.0，则认为存在中性选择；如果Ka/Ks<1.0，则认为存在净化选择效应。公式T = Ks/2λ (λ = 1.5×10⁻⁸)用于计算HAKs家族基因的分化时间^[54]。

1.4 HAKs家族基因的多序列比对和系统发育分析

Clustal W软件用于比对拟南芥和油菜中HAKs家族基因的蛋白质序列，然后使用MEGA7.0软件(http://www.megasoftwar-e.net/)通过邻接法(NJ)构建系统发育树^[55]。为了获得更可靠的分支聚类，我们将Bootstrap值设置为1000，并将其余参数设置为默认值^[56]。

1.5 HAKs家族基因的保守基序/域和基因结构分析

为了研究拟南芥和芸薹属作物中HAKs家族基因的结构差异，将它们的蛋白质序列提交给在线软件MEME 4.12.0 (http://memes-uite.org/tools/meme)以预测HAKs家族基因的保守结构元件^[57]，最大基序数设置为10。将拟南芥和甘蓝型油菜HAKs家族成员的全长基因组DNA (gDNA)和CDS序列提交到在线软件Gene Structure Display Server (GSDS) 2.0 (http://gsds.cbi.pku.edu.cn/)中，预测并绘制HAKs家族基因的外显子—内含子结构示意图^[58]。

1.6 HAKs家族基因的启动子调控元件分析

为了鉴定拟南芥和油菜HAKs家族基因启动子中可能的顺式作用调控元件，我们从拟南芥数据库TAIR (https://www.arabidopsis.org/)和油菜基因组数据库(http://www.genoscope.cns.fr/brassicana-pus/)中，下载HAKs同源基因起始密码子上游2.0 kb的DNA序列^[59]，并将该序列提交至PLACE v. 30.0 (http://www.dna.affr-c.go.jp/PLACE/)以识别可以与启动子区域结合的顺式作用元件^[60]。利用GSDS2.0 (http://gsds.gao-lab.org/)在线软件进行可视化。

1.7 植物材料培养及处理

为了鉴定甘蓝型油菜BnaHAKs与油菜响应非生物胁迫的关系，将甘蓝型油菜Westar在湿纱布上发芽5天，然后选择长势相同的幼苗，转移到10 L Hoagland营养液(1.0 mmol/L KH₂PO₄、5.0 mmol/L KNO₃、5.0 mmol/L Ca(NO₃)₂·4H₂O、2.0 mmol/L MgSO₄·7H₂O、9.0 μmol/L MnCl₂·4H₂O、0.80 μmol/L ZnSO₄·7H₂O、0.30 μmol/L CuSO₄·5H₂O、0.10 μmol/L Na₂MoO₄·2H₂O和46 μmol/L H₃BO₃)中，于光照室中培养，光照强度为200 μmol/(m²·s)，昼/夜温度25℃/ 22℃，光照周期16 h /8 h，相对湿度70%，每5天更换1次营养液。

为了进一步分析不同营养胁迫下BnaHAKs的表达模式，设定了4种处理。在低钾处理中，将萌发一致的种子进行处理，前期用霍格兰营养液进行培养，长至两片真叶后进行低钾(0.03 mmol/L)和对照(正常钾，6.0 mmol/L)处理。低钾处理3天后取样。在盐胁迫处理中，将长势一致的油菜幼苗在无NaCl溶液中培养10 天，然后转移到200 mmol/L NaCl溶液中生长10 天。在无机磷酸盐(Pi)饥饿处理中，先将长势一致的种子萌发5 天龄油菜幼苗在1.0 mmol/L Pi (KH₂PO₄)溶液中培养10天，然后在250 μmol/L (KH₂PO₄)溶液中生长，直至取样；在镉毒性处理中，将种子萌发后的幼苗在无镉溶液中水培10 天，然后转入10 μmol/L CdCl₂溶液中培养，直至取样。

此外，在低钾条件下，还使用两个甘蓝型油菜 “H280”(耐低钾基因型)和 “L49”(低钾敏感基因型)品系用于后续的试验。上述新鲜油菜幼苗的地上部和根分别取样，用液氮将样品研磨之后存储于超低温冰箱(−80℃)备用。每个处理进行3个独立重复，每个处理对20株油菜植株取样。利用上海美吉生物医药科技有限公司Illumina高通量测序平台进行转录组测序。

1.8 不同营养胁迫下BnaHAKs家族基因的转录及基因共表达网络分析

为了分析不同样品/组之间的差异，每个基因的表达水平按TPM法计算，RSEM法量化基因丰度^[61]。构建基因共表达网络时，针对差异表达基因(P≤0.05; |log² (FC)| ≥ 1)使用Rstudio软件计算相关性指数，以识别基因相互作用并找到核心基因，对于每对基因，皮尔森相关系数的阈值按照默认设置(http://plantgrn.noble.org/DeGNServer/Analysis.jsp)，随后通过CYTOSCAPE 3.2.1构建基因共表达网络图(http://www.cytoscape.org/)^[62]。

1.9 低钾条件下实时荧光定量PCR (RT-qPCR)分析

用RNase-free DNase I从RNA样品中去除基因组DNA后，用PrimeScript^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Shiga, Japan)将总RNA作为模板进行cDNA合成，随后使用SYBR FAST qPCR Master Mix (南京，诺唯赞)试剂盒在Roche Light Cycler480 荧光定量PCR仪进行RT-qPCR测定，采用 2^−ΔΔC T 的方法^[63]。RT-qPCR反应条件：95ºC 3 min，40循环(95ºC 10 s，60ºC 3 s)。使用已报道的BnActin和Tubulin作为参考基因^[64]，将表达数据归一化，每个样品包括3个独立的生物学重复。

1.10 亚细胞定位分析

为了确定BnaA7.HAK5蛋白的亚细胞定位，构建了BnaA7.HAK5与表达载体GFP的重组载体BnaA7.HAK5-GFP，并将其转化至4周龄的烟草叶片中，取注射部位附近生长2天的烟草叶片浸泡于FM4-64染料中，随后用激光扫描共聚焦显微镜(Leica TCSSP8 DMI8 LASX)观察荧光，发射波长分别为734 nm，激发波长为558 nm^[65−66]。

1.11 矿质元素含量的测定

选择长势一致的幼苗，用去离子水冲洗干净，放置65°C烘箱烘干至恒重并记录重量，每个样品包括5个生物学重复。随后将干燥后的地上部和根部分别转移到200°C的HNO₃/HClO₄混合物中直至消化完成。使用电感耦合等离子质谱仪(ICP-MS；NexIONTM350X，PerkinElmer)测定K⁺含量^[67]。

2. 结果与分析

2.1 HAKs家族基因的全基因组鉴定

为了鉴定芸薹属作物中HAKs的家族成员，以拟南芥中HAKs蛋白质序列作为查询对象，结果表明，甘蓝型油菜中共鉴定到40个HAKs家族成员；白菜和甘蓝中分别鉴定到21和23个HAKs同源基因(表1)。此外，Brassica napus中HAKs家族成员的数量与B. rapa和B. oleracea物种中HAKs基因的总和基本一致(表1)。因此，可以推测大部分HAKs家族基因在B. rapa和B. oleracea的自发杂交过程中保留了下来。其中，HAKs家族成员中HAK5s、HAK7s、HAK8s、HAK10s、HAK11s、HAK12s和HAK13s均具有2个同源基因；HAK2s、HAK3s均具有3个同源基因；HAK4s具有3个同源基因；HAK1s和HAK6s均具有4个同源基因；HAK9s具有10个同源基因，数量的差异解释了HAKs家族基因在油菜异源多倍体形成过程中的差异扩展模式。

表 1 拟南芥和芸薹属作物中HAKs家族基因的拷贝数

Table 1. Gene copy numbers of HAKs family in Arabidopsis and Brassica crops

基因名称 Gene name	拟南芥 Arabidopsis thaliana (125 Mb)	白菜 Brassica rapa (465 Mb)	甘蓝 Brassica oleracea (485 Mb)	甘蓝型油菜 Brassica napus
HAK1	1	3	1	4
HAK2	1	1	2	3
HAK3	1	2	2	3
HAK4	1	2	2	2
HAK5	1	1	1	2
HAK6	1	1	2	4
HAK7	1	1	2	2
HAK8	1	1	2	2
HAK9	1	3	4	10
HAK10	1	2	1	2
HAK11	1	2	2	2
HAK12	1	1	1	2
HAK13	1	1	1	2
总计 Total	13	21	23	40

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2.2 HAKs家族基因的分子特征

为了解甘蓝型油菜中HAKs家族基因的分子特征，本研究使用ExPASy计算40个BnaHAKs蛋白的物理和化学参数。结果表明，HAKs蛋白质大小在402～864个氨基酸之间变化，其中BnaC7.HAK9c的蛋白序列最短(402个氨基酸)，BnaA3.HAK7和BnaC3.HAK7的蛋白序列最长(864个氨基酸) (附表1)。HAKs家族基因AI值在87.1～114.98之间变化(附表2)。HAKs家族蛋白中HAK2s、HAK3s、HAK4s、HAK6s、HAK7s、HAK8s、HAK12s和HAK13s的IIs>40.0，表现出较弱的蛋白质稳定性；而HAK1s、HAK5s、HAK9s和HAK11s的IIs<40.0，表现出较强的蛋白质稳定性。此外，BnaA2.HAK8和BnaC2.HAK8平均亲水系数为负值，属于亲水性蛋白，其余HAKs家族基因的GRAVY值是正的，属于疏水性蛋白质(附表2)。

为了进一步确定BnaHAKs的跨膜拓扑结构，我们使用TMHMM工具对其跨膜结构进行表征，发现40个BnaHAKs家族成员具有9～15个跨膜结构域(附图1、附表2)。将HAKs家族基因的氨基酸序列提交给WoLF PSORT以预测它们的亚细胞定位，结果表明，HAKs家族基因广泛的分布在液泡、内质网、高尔基体、叶绿体、细胞质，其中大部分HAKs家族成员位于细胞膜上(附表2)，说明这些基因与K⁺吸收和植物体内K⁺平衡有关。

基因家族主要由串联复制、节段复制、全基因组复制、复制转座等组成^[68]。比较基因组学表明拟南芥基因组被分为24个祖先十字花科植物块，标记为A-X^[47]。为更好地理解BnaHAKs基因的扩展方式，本研究对其重复事件进行了探讨。40个HAK1s分别位于I (HAK1s)、J (HAK2s和HAK11s)、F (HAK3s)、U (HAK4s，HAK9s和HAK13s)、T (HAK5s)、E (HAK6s)、R (HAK7s和HAK8s)、B (HAK10s)以及D (HAK12s) (附表1)。从HAKs家族成员的基因组分布来看，我们发现这些基因的家族扩展主要是由于全基因组重复和节段重复。

在遗传学中，Ka/Ks表示两个蛋白编码基因的非同义替换率(Ka)和同义替换率(Ks)之间的比例。为了解甘蓝型油菜HAKs家族基因的进化选择效应，我们使用HAKs家族基因之间的同源基因对计算Ks、Ka和Ka/Ks值(附表1)。结果表明，HAKs的Ka值范围为0.01～0.09，平均值为0.46；Ks值范围为0.36～0.79，平均值为0.54。40个BnaHAKs家族基因的Ka/Ks值均小于1.0 (附表1)，因此，认为这些基因在油菜中进行了纯化选择，更有利于基因功能的保留^[69]。

2.3 HAKs家族基因的系统发育分析

为进一步分析拟南芥和甘蓝型油菜HAKs蛋白之间的分子进化和系统发育关系，我们构建了系统发育树(图1)。甘蓝型油菜HAKs家族成员和拟南芥的HAKs成员均匀的聚类在进化树的各个分支上，聚类在一起的BnaHAKs和AtHAKs蛋白序列高度同源，表明甘蓝型油菜BnaHAKs在进化过程中没有发生大规模的变异(图1)。保守基序是具有特定功能的蛋白质结构，每个基序都有其特有的氨基酸序列来执行其功能。本研究使用MEME对甘蓝型油菜HAKs家族基因的保守基序进行预测和分析，结果表明BnaA9.HAK3缺少motif1、5、8、9，BnaC2.HAK6缺少motif9，其它38个HAKs家族基因成员均含有预测的10个保守基序，表明这些基因在进化中比较保守(图1)。基因结构分析表明，BnaA2.HAK8外显子最多，达20个；其次为BnaC2.HAK8，外显子数目为19个；而其他成员的外显子数目在5～18，变异较大。而在所有的HAKs家族成员中外显子和内含子数目相近，表明甘蓝型油菜HAKs在基因进化过程中相对保守。以上结果说明，HAKs家族基因在氨基酸残基进化方面是非常保守的，表明它们在基因功能或者结构方面具有非常重要的作用。

图 1 拟南芥和甘蓝型油菜中HAKs家族基因系统发育分析

注：(a)保守基序，(b)基因结构。图a为BnaHAKs蛋白保守的10个基序(motif)及其分布位置，不同颜色的方框表示不同的保守基序(基序1～10)，灰色线条表示未检测保守基序的HAKs蛋白区域。图b为BnaHAKs基因结构分布图。

Figure 1. Phylogenetic analysis of HAKs family genes in Arabidopsis and Brassica napus

Note: (a) the conserved motifs, (b) gene structure. Figure a shows the 10 motifs (motifs) conserved in BnaHAKs proteins and their distribution locations, different colored boxes indicate different conserved motifs (motifs 1−10), and gray lines indicate the HAK protein regions where conserved motifs were not detected. Figure b shows the distribution of BnaHAKs gene structure.

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2.4 HAKs家族基因的染色体定位

鉴定出油菜中40个HAKs家族成员通过物理映射，结果如图2所示，共有38个BnaHAKs定位在甘蓝型油菜的16条染色体上(图2)。18个BnaHAKs定位在A亚基因组，20个BnaHAKs定位在C亚基因组。其中A4、A9、C8和C9的染色体上各有1个HAKs；A2、A5、A7、A8、C2和C6的染色体上各有2个HAKs；在C3和C7的染色体上各3个HAKs；A1、A3、C1和C4的染色体上各有4个HAKs (图2，附表1、附表2)。

图 2 拟南芥和油菜HAKs家族成员的染色体定位

Figure 2. Chromosomal location of HAKs family members in Arabidopsis and Brassica napus

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2.5 HAKs家族基因的顺式作用调控元件分析

为了鉴定HAKs家族基因的核心转录因子，本研究将HAKs家族基因起始密码子(ATG)上游2.0 kb基因组序列提交给PLACE v. 30.0，以预测可能的顺式作用调控元件(图3)。结果表明，共有107种类型的CRE (3717个顺式作用元件)能结合到BnaHAKs家族基因启动子，说明BnaHAKs家族基因的转录调控是一个复杂的网络调控系统。在3717个顺式元件中，选择富集程度最高的5种顺式作用元件作图(图3)。结果表明，除了常见的TATA-box、CAAT-box和G-box之外，本研究还发现MYB和激素响应顺式作用调控元件ABRE在启动子区域高度富集(图3)，表明HAKs家族基因有可能通过不同的激素和非生物胁迫响应途径调控油菜的生长发育以及非生物胁迫抗性反应。

图 3 HAKs家族基因的顺式作用调控元件富集分析

Figure 3. Enrichment analysis of cis-acting regulatory elements of HAKs family genes

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2.6 HAKs家族基因共线性分析

为了进一步揭示物种内部以及与祖先物种间的进化线性关系，本研究利用比较基因组学的方法分析了甘蓝型油菜物种内部以及甘蓝型油菜与拟南芥、白菜及甘蓝之间的同源关系。结果表明，甘蓝型油菜物种内共检测到42对HAKs共线性基因(图4)。在物种间拟南芥HAKs基因家族的成员均能映射到甘蓝型油菜的染色体上(图5)；且甘蓝型油菜HAKs基因家族40个成员，在白菜中有25个同源基因，在甘蓝中也有25个同源基因(图5)。

图 4 甘蓝型油菜HAKs基因家族中AC亚基因组共线性分析

Figure 4. Collinearity analysis of AC subgenome in HAKs gene families in Brassica napus

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图 5 HAKs家族基因在拟南芥、白菜、甘蓝以及甘蓝型油菜之间的共线性分析

Figure 5. Collinearity analysis of HAKs family genes in Arabidopsis, B. rapa, B. oleracea, and B. napus

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2.7 不同养分胁迫下BnaHAKs的转录分析

为了探究甘蓝型油菜HAKs家族基因在K⁺吸收和转运方面的作用，本研究分析了正常钾和低钾胁迫下的转录情况，结果表明无论是在地上部还是在根部，大部分HAKs家族成员的表达水平被低钾胁迫显著诱导，尤其是根部BnaA7.HAK5的表达丰度被低钾胁迫显著上调3.9倍，地上部BnaA5.HAK1和BnaC4.HAK1的表达水平分别被低钾胁迫显著上调3.3和2.2倍(图6a)，表明甘蓝型油菜HAKs家族基因在低钾胁迫下通过上调表达发挥功能，促使植物正常生长。在多倍体甘蓝型油菜中，多拷贝基因家族很常见，核心基因的鉴定是了解重要农艺性状分子机制的关键先决条件^[51−52]。因此，核心基因的鉴定将有助于我们全面了解植物对非生物胁迫的适应性。为了识别HAKs家族基因中的核心成员，构建了基因共表达网络图，结果表明，HAKs家族中BnaA7.HAK5被确定为核心成员，BnaA5.HAK11、BnaC3.HAK1、BnaC4.HAK1、BnaC6.HAK5以及BnaA3.HAK1可能在低钾胁迫条件下发挥次要作用(图7a)，以上结果表明，BnaA7.HAK5可能在植物响应低钾胁迫过程中发挥关键作用。

图 6 不同营养胁迫下甘蓝型油菜BnaHAKs家族基因的差异表达谱热图

注：(a)低钾处理，(b)盐胁迫，(c)低磷处理，(d)镉处理。颜色代表相对表达水平从高(红色)到低(蓝色)。图a～d中，S、R分别表示地上部、根部。HK，正常钾浓度(6 mmol/L K₂HPO₄)；LK，低钾胁迫(0.03 mmol/L K₂HPO₄)；NaCl，盐胁迫(200 mmol/L NaCl)；−Pi，低磷处理(250 μmol/L K₂HPO₄)；+Cd：镉胁迫(10 μmol/L CdCl₂)。

Figure 6. Heat maps of differential expression of BnaHAKs family genes in Brassica napus under different nutrient stresses

Note: (a) Low K treatment, (b) Salt stress treatment, (c) Low Pi treatment, (d) Cadmium toxicity treatment. The color scales represent relative expression levels from high (red color) to low (blue color). Figure a−d: S and R indicate shoots and roots, respectively. HK, normal potassium concentration (6 mmol/L K₂HPO₄); LK, low potassium stress (0.03 mmol/L K₂HPO₄); NaCl, salt stress (200 mmol/L NaCl); −Pi, low phosphorus treatment (250 μmol/L K₂HPO₄); +Cd, cadmium stress (10 μmol/L CdCl₂).

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图 7 不同处理下甘蓝型油菜BnaHAKs基因家族差异共表达网络分析

注：圆圈及其颜色表示该基因在网络中的作用，圆圈越大，颜色越深，表明该基因在该网络中的作用越大；圆圈之间的线条粗细表示基因间的互作程度，线条越粗表示基因间的互作程度越大。

Figure 7. Network analysis of differential co-expression of BnaHAKs gene family in Brassica napus under different treatments

Note: The circles and their colors indicate the role of the gene in the network, the larger the circle, the darker the color, indicating the greater the role of the gene in that network. The thickness of the lines between the circles indicates the degree of interaction between genes, the thicker the line, the greater the degree of interaction between genes.

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为了探究BnaHAKs在其他营养胁迫下的作用，本研究分析了不同营养胁迫下差异基因的转录响应(图6)。在盐胁迫下，共鉴定出19个在地上部或根部响应盐胁迫的差异表达基因(图6b)。其中包括7个BnaHAK9s、2个BnaHAK11s、2个BnaHAK5s、3个BnaHAK6s、2个BnaHAK1s以及1个BnaHAK2和BnaHAK8。我们发现BnaA5.HAK1、BnaA7.HAK5、BnaC6.HAK5、BnaC4.HAK1的表达量在盐处理后的根部显著升高，而BnaC2.HAK8、Bna.HAK9s在盐处理后的根部显著下调(图6b)。为了确定在盐胁迫响应中起主导作用的核心成员，对BnaHAKs进行共表达网络分析。结果表明，BnaC6.HAK5和BnaA7.HAK5可能在油菜适应盐胁迫方面发挥关键作用(图7b)。本研究又在磷酸盐胁迫下分析了BnaHAKs的转录组数据，共鉴定出14个差异表达基因。在这些差异表达基因中，参与非生物胁迫的BnaHAK5s在根部表达量上调最为明显(图6c)，基因共表达网络分析表明，BnaC6.HAK5和BnaA7.HAK5可能在油菜植株对低磷胁迫的响应中起核心作用(图7c)。在镉毒性下，在地上部或根系中共鉴定出8个BnaHAKs (图6d)。值得注意的是，镉处理后的根中大多数基因表达下调，尤其是BnaA7.HAK5的表达显著下调(图6d)。

2.8 油菜BnaA7.HAK5调控低钾胁迫抗性的功能初步解析

在实验室的前期工作中，已从259份自然油菜品系中筛选出耐低钾基因型品系“H280”和低钾敏感基因型油菜品系“L49”^[67]。本研究先对油菜品系“H280”(耐低钾基因型)和“L49”(低钾敏感基因型)在5个钾浓度下的生长响应进行探究，发现两个品系的地上部整体长势呈现出相同的趋势，随着钾浓度的降低，植株矮小和叶片黄化程度逐渐加强(图8a)，尤其是在低钾(0.03 mmol/L)浓度下，“H280”油菜品系和“L49”油菜品系的地上部整体长势呈现显著差异(图8a)，因此最终确定6.0 mmol/L为正常钾浓度，0.03 mmol/L为低钾浓度进行后续的试验。

图 8 油菜BnaA7.HAK5调控低钾胁迫抗性的功能初步解析

注：(a～d)正常钾和低钾条件下，“H280”和“L49”生长表型、K⁺含量以及基因表达(a)，“H280”和“L49”在6.0、3.0、1.0、0.3、0.03 mmol/L (由左至右) 钾浓度条件下的地上部整体长势；(b)，各叶片的形态差异；(c)，根部K⁺含量. High K，钾离子浓度为6.0 mmol/L，Low K，钾离子浓度为0.03 mmol/L；(d)，根部BnaA7.HAK5表达量分析；(e) BnaA7.HAK5亚细胞定位分析。

Figure 8. Preliminary functional analysis of the regulation of low K stress resistance by BnaA7.HAK5 in Brassica napus

Note: (a−d) Growth phenotypes, K⁺ content and gene expression of “H280” and “L49” under normal and low K stress conditions. (a), Growth phenotypes of “H280” and “L49” under different K concentrations of 6.0 mmol/L, 3.0 mmol/L, 1.0 mmol/L, 0.3 mmol/L, 0.03 mmol/L (from left to right); (b), morphological differences of each leaf; (c), K⁺ contentration of roots. High K, K concentrations of 6.0 mmol/L, Low K, K concentrations of 0.03 mmol/L; (d), BnaA7.HAK5 expression analysis of roots; (e) subcellular localization analysis of BnaA7.HAK5.

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低钾胁迫会使植株地上部各叶片形态发生差异，并导致叶片黄化。与正常钾处理相比，低钾处理后“L49”叶片衰老程度大于“H280”，且“L49”油菜品系的新叶显著小于“H280”(图8b)随后对正常钾和低钾条件下水培的“H280”和“L49”油菜品系根部的离子组和BnaA7.HAK5基因表达量分析表明，在低钾条件下，“H280”油菜品系中K⁺含量和BnaA7.HK5表达量显著高于“L49”油菜品系(图8c,d)。为研究BnaA7.HAK5在细胞中的定位，本研究将BnaA7.HAK5与表达载体GFP连接进行亚细胞定位^[65]，结果表明BnaA7.HAK5-GFP主要位于细胞质膜上(图8e)。

3. 讨论

已有研究表明，HAK/KUP/KT(HAK)家族是植物中最大的K⁺转运蛋白家族，其作用是负责钾离子的吸收和转运以及植物生长发育、气孔运动、耐盐性等^[14−15]。然而，目前对甘蓝型油菜中HAKs的系统性研究较少。本研究鉴定了甘蓝型油菜HAK家族基因及其系统发育关系、基因结构、保守基序、染色体定位、顺式作用元件。此外，本研究描述了BnaHAKs在不同养分胁迫条件下的差异表达谱以及低钾胁迫下核心基因的初步解析。BnaHAKs的全面鉴定为进一步深入研究低钾胁迫奠定了基础。

3.1 综合生物信息学分析全面揭示了BnaHAKs的分子特征

通过对芸薹属作物和拟南芥全基因组HAKs基因家族的鉴定发现，甘蓝型油菜中40个BnaHAKs均有2～10个拷贝，说明它们在进化过程中并没有基因丢失的现象发生^[70]。HAKs家族在植物中普遍存在，如拟南芥中有13个AtHAKs^[71]，梨中有21个PbHAKs^[72]，马铃薯中有16个StHAKs^[73]，水稻中有27个OsHAKs^[74]。油菜中HAK基因家族比其他植物种中成员更多，这可能与油菜基因组发生的全基因组复制和后续进化有关^[75]。植物HAK家族可划分为5个系统进化簇^[76]，然而一些植物种中只有4个进化簇，如石榴、拟南芥和葡萄的HAK/KUP/KT家族基因均有4个簇(Ⅰ～Ⅳ)^[77−79]。系统发育分析表明，甘蓝型油菜可分为4个簇(Cluster I～Cluster V，缺少Cluster IV，图1)，导致这种现象的原因可能是在进化过程中发生了丢失。这也支持了第4组在十字花科植物中不存在的结论^[76]。基因结构显示，BnaHAKs的基因组序列长度在2.8～11 kb，含有4～19个内含子(图1)，内含子长度的差异可能在BnaHAKs功能多样化中起作用。

顺式作用元件是转录调控的分子开关，对基因表达调控起到关键作用。已有研究表明，HAKs基因对各种胁迫类型和植物激素非常敏感，并积极调节植物的胁迫反应^[80−82]。转录因子预测显示，许多与植物生长发育、胁迫以及植物激素反应相关的顺式作用元件广泛分布在HAKs基因的启动子区域，表明BnaHAKs可能参与非生物胁迫和植物激素应答相关的反应(图3)。同时前人也通过实验表明，不同物种中启动子区域的顺式作用元件与HAKs结合均可诱导HAKs的表达^[83]，通过分析HAKs家族基因顺式作用元件发现，BnaHAKs家族成员启动子区域均含有大量的MYB转录因子。已有研究表明，MYB作为上游转录因子调控HAK5的表达。例如，在拟南芥中，Feng等^[84]结合启动子序列分析，体外烟草瞬时表达实验，电泳迁移率位移试验(EMSA)，及体内染色质免疫沉淀分析证明MYB77位于HAK5的上游，且MYB77直接结合HAK5的启动子区域促进HAK5的表达。基于转录组分析，发现在低钾胁迫下，BnaC7.MYB77的表达量在抗低钾品系“H280”的根部显著高于低钾敏感品系“L49”，说明BnaC7.MYB77可能正调控BnaA7.HAK5的表达，推测MYB77-HAK可能是高效耐钾油菜品系抵抗低钾胁迫的关键分子通路。本研究继续分析了盐胁迫、低磷和镉毒害胁迫下MYB家族成员的转录情况，发现MYB家族成员在盐胁迫、低磷以及镉毒害胁迫的根部表达量普遍上调(附图2)，且HAK5在盐胁迫、低磷胁迫下的根部表达量与对照组相比存在显著差异(图6)。因此本研究推测MYB转录因子参与调控HAKs的表达。

3.2 BnaHAKs家族成员在响应不同逆境胁迫中具有复杂性

自第一个HAK从大麦中克隆出来，已经在各种植物种中发现了许多同源物并进行了功能分析^[85−88]。HAK主要参与高亲和力K⁺吸收和转运。例如拟南芥AtHAK1^[89]、大麦HvHAKs^[23]、水稻OsHAK1^[33]受低钾诱导，参与K⁺吸收，维持细胞中的K⁺平衡。同样，基于低钾胁迫下的差异表达分析可以发现，低钾处理显著诱导了BnaHAKs在地上部和根中的表达水平，其中根部BnaA7.HAK5的上调水平最高(图6a)。这与前人的研究结果相符合，拟南芥中发现受低钾胁迫转录水平最为显著的基因是HAK5^[90]。另外，大麦中的HvHAK1^[23]，桑葚MaHAK^[91]以及西红柿中的LeHAK5^[92]等基因转录水平受低钾胁迫的诱导而发生显著性变化。

盐胁迫已成为制约作物产量和品质的非生物胁迫之一。研究表明，维持细胞内Na⁺/K⁺的稳态或降低Na⁺/K⁺的值是植物耐盐性的关键^[93]。在本研究中发现大多数BnaHAKs在盐处理后的根中显著上调(图6-b)，与期的结果一致。前人研究结果表明，超表达OsHAK5可以增强转基因烟草的耐盐性，导致这种情况是因为OsHAK5过表达增加了细胞中K⁺的含量^[94]。在玉米中，ZmHAK4在根系中优先表达，其通过降低根系中Na⁺的含量，以提高玉米的耐盐性^[95]。与玉米ZmHAK4一样，水稻OsHAK16^[32]和OsHAK11^[96]也具有耐盐性，说明HAKs家族成员在植物耐盐性中发挥着重要作用。此外，由于对BnaHAKs的转录水平有了更广泛的研究，还发现了BnaHAKs家族成员除了受低钾和盐胁迫处理的影响外，其转录水平在许多养分胁迫下均受到影响。本研究转录组分析结果显示，在不同养分胁迫中，BnaHAKs的转录水平有着不完全相同的表达趋势。其中BnaA7.HAK5在低钾、盐胁迫和低磷处理下有着相同的表达趋势，但是在镉处理的根部有着相反的趋势(图6)，意味着甘蓝型油菜BnaHAKs家族成员在响应不同逆境胁迫中具有复杂性。有趣的是，由于Cs⁺和K⁺之间存在相似性，HAKs家族成员可以介导高亲和性Cs⁺的吸收^[97]，但是BnaHAKs家族成员在低磷和镉毒性的条件下转录水平发生改变，关于其家族成员是否参与低磷胁迫和镉毒性的响应尚未有报道。

3.3 BnaA7.HAK5可能在油菜响应低钾胁迫抗性反应中发挥核心作用

据报道，高亲和性K⁺转运蛋白HAK5在调节植物钾营养过程中具有重要的作用^[98−99]。在水稻中，OsHAK5作为K⁺转运体参与非生物胁迫下K⁺的吸收和转运。研究表明，OsHAK5植株在钾离子浓度为0.01 mmol/L的酵母培养基上正常生长，敲除OsHAK5基因的水稻植株根系中钾离子的含量受到严重的影响，而OsHAK5超表达植株促进根系钾离子的吸收^[100−101]；玉米ZmHAK5也是一种高亲和力钾离子转运蛋白，ZmHAK5突变体植株的钾离子吸收能力较弱，而超表达植株钾离子吸收能力和水稻的超表达植株一样，具有较强的K⁺吸收能力^[102]；同时，AtHAK5^[16]也被证实是拟南芥应对低钾胁迫反应的标志基因，在拟南芥的根中，受到低钾胁迫时AtHAK5转录水平增加，诱导K⁺吸收。分析低钾胁迫下的转录水平可以发现，BnaA7.HAK5的表达丰度显著上调(图6a)。值得注意的是，通过对BnaA7.HAK5的定量分析，发现在低钾条件下，BnaA7.HAK5的表达量仍然显著上调，且在“H280”和“L49”油菜品系间的根部呈现显著差异(图8c)，亚细胞定位说明BnaA7.HAK5定位在细胞质膜上(图8e)。表明BnaA7.HAK5可能是造成两个油菜品种对低钾胁迫有不同抗性的原因之一，但是BnaA7.HAK5响应油菜低钾胁迫的具体机制还有待进一步研究。

4. 结论

本研究在甘蓝型油菜中共鉴定到40个HAKs家族成员。基于转录组数据和基因共表达网络结果表明，BnaA7.HAK5基因响应低钾或盐等养分胁迫。然而，要确认这些核心基因的功能还需要更进一步的研究。今后将继续深入对HAKs家族基因进化的研究，并为进一步揭示油菜抗营养胁迫的转录调控机制提供有价值的候选基因。

附图 1 HAKs家族基因跨膜特性

Supplementary figure 1. Transmembrane characteristics of HAKs family genes

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1 拟南芥和油菜中HAKs家族基因的理化性质

1. Physicochemical properties of HAKs family genes in Arabidopsis and Brassica napus

基因ID Gene ID	基因名称 Gene name	不稳定系数 Instability index	脂肪指数 Aliphatic index	平均亲水系数 Average hydrophobic index	跨膜结构域 Trans-membrane domains	亚细胞定位 Subcellular localization
AT2G30070	AtHAK1	39.39	105.72	0.483	13	Endoplamic reticulum, mitochondrion, nucleus, plasmolemma, vacuole
BnaA03g13690D	BnaA3.HAK1	37.36	107.79	0.485	13	Chloroplast, endoplamic reticulum, plasmolemma
BnaA05g12300D	BnaA5.HAK1	36.15	105.97	0.459	13	Chloroplast, cytoplasm, endoplamic reticulum, plasmolemma
BnaC03g16580D	BnaC3.HAK1	36.68	109.32	0.499	13	Chloroplast, endoplamic reticulum, plasmolemma
BnaC04g14750D	BnaC4.HAK1	35.74	106.81	0.48	12	Endoplamic reticulum, plasmolemma, vacuole
AT2G40540	AtHAK2	45.54	108.12	0.349	12	Chloroplast, endoplamic reticulum, plasmolemma
BnaA04g23330D	BnaA4.HAK2	42.75	108.36	0.349	12	Chloroplast, endoplamic reticulum, plasmolemma, vacuole
BnaC04g01430D	BnaC4.HAK2a	45.39	101.44	0.133	13	Chloroplast, endoplamic reticulum, plasmolemma, vacuole
BnaC04g47240D	BnaC4.HAK2b	42.85	107.84	0.342	12	Chloroplast, endoplamic reticulum, plasmolemma, vacuole
AT3G02050	AtHAK3	43.85	107.16	0.434	14	Endoplamic reticulum, plasmolemma
BnaA01g13320D	BnaA1.HAK3	43.79	106.19	0.427	12	Plasmolemma
BnaA09g21950D	BnaA9.HAK3	44.12	107.45	0.557	11	Endoplamic reticulum, plasmolemma, vacuole
BnaC01g15360D	BnaC1.HAK3	43.31	106.31	0.438	10	Endoplamic reticulum, plasmolemma, vacuole
AT4G23640	AtHAK4	43.85	107.16	0.434	14	Endoplamic reticulum, golgi apparatus, plasmolemma
BnaC09g24170D	BnaC9.HAK4	41.62	111.74	0.642	15	Endoplamic reticulum, golgi apparatus, plasmolemma
BnaCnng05490D	BnaCn.HAK4	44.9	107.83	0.391	13	Plasmolemma
AT4G13420	AtHAK5	29.71	101.38	0.222	12	Endoplamic reticulum, plasmolemma
BnaA07g16500D	BnaA7.HAK5	28.38	100.43	0.194	10	Endoplamic reticulum, plasmolemma
BnaC06g15440D	BnaC6.HAK5	28.48	100.04	0.192	12	Endoplamic reticulum, plasmolemma
AT1G70300	AtHAK6	46.34	105.81	0.298	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaA02g14820D	BnaA2.HAK6	47.18	111.08	0.445	12	Plasmolemma, vacuole
BnaA07g38760D	BnaA7.HAK6	42.73	110.1	0.414	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaC02g19780D	BnaC2.HAK6	45.08	106.85	0.325	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaC06g31400D	BnaC6.HAK6	45.3	104.37	0.251	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
AT5G09400	AtHAK7	41.87	105.59	0.297	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaA03g02700D	BnaA3.HAK7	43.61	104.31	0.268	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole

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续附表 1　Table S1 continued
基因ID Gene ID	基因名称 Gene name	不稳定系数 Instability index	脂肪指数 Aliphatic index	平均亲水系数 Average hydrophobic index	跨膜结构域 Trans-membrane domains	亚细胞定位 Subcellular localization
BnaC03g03790D	BnaC3.HAK7	43.03	104.76	0.271	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
AT5G14880	AtHAK8	38.5	107.41	0.306	13	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaA02g02340D	BnaA2.HAK8	43.03	87.1	-0.145	13	Endoplamic reticulum, mitochondrion, plasmolemma
BnaC02g05800D	BnaC2.HAK8	42.98	87.03	-0.137	15	Endoplamic reticulum, mitochondrion, nucleus, plasmolemma
AT4G19960	AtHAK9	34.64	105.01	0.298	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA01g10310D	BnaA1.HAK9	35.67	105.7	0.287	13	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA03g44320D	BnaA3.HAK9a	33.96	106.1	0.259	13	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA03g44330D	BnaA3.HAK9b	32.73	106.58	0.273	13	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA08g30510D	BnaA8.HAK9b	38.04	107.57	0.278	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaC01g41320D	BnaC1.HAK9	36.57	106.92	0.342	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaC03g76940D	BnaC3.HAK9	37.14	107.5	0.296	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaC07g36080D	BnaC7.HAK9a	38.62	105.78	0.23	11	Cytoplasm, endoplamic reticulum, nucleus, plasmolemma, vacuole
BnaC07g36130D	BnaC7.HAK9b	35.13	105.73	0.284	13	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaC07g36140D	BnaC7.HAK9c	29.93	114.98	0.504	9	Chloroplast, cytoplasm, endoplamic reticulum, plasmolemma, vacuole
BnaCnng46720D	BnaCn.HAK9	30.02	107.85	0.318	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
AT1G31120	AtHAK10	40.19	103.54	0.3	11	Endoplamic reticulum, plasmolemma
BnaA08g08020D	BnaA8.HAK10	40.07	106.43	0.341	11	Endoplamic reticulum, plasmolemma
BnaC08g09300D	BnaC8.HAK10	37.98	103.44	0.289	11	Endoplamic reticulum, plasmolemma
AT2G35060	AtHAK11	37.2	105.79	0.322	11	Endoplamic reticulum, plasmolemma, vacuole
BnaA05g08850D	BnaA5.HAK11	38.04	106.1	0.35	13	Endoplamic reticulum, plasmolemma, vacuole
BnaC04g10260D	BnaC4.HAK11	38.26	109.62	0.412	14	Endoplamic reticulum, plasmolemma, vacuole
AT1G60160	AtHAK12	42.72	108.33	0.376	13	Endoplamic reticulum, plasmolemma
BnaA01g22220D	BnaA1.HAK12	42.3	108.28	0.395	13	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaC01g43090D	BnaC1.HAK12	40.31	108.73	0.395	13	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
AT4G33530	AtHAK13	39.94	109.43	0.331	11	Endoplamic reticulum, golgi apparatus, mitochondrion, plasmolemma, vacuole
BnaA01g03380D	BnaA1.HAK13	40.87	108.07	0.329	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaC01g04660D	BnaC1.HAK13	42.84	108.72	0.333	11	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole

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2 拟南芥和油菜中HAKs家族基因的分子特征

2. Molecular characteristics of HAKs family genes in Arabidopsis and Brassica napus

基因ID Gene ID	基因名称 Gene name	区块 Block	蛋白长度 Protein length (aa)	编码区长度 Coding sequences length (bp)	DNA全长 DNA length	外显子/内含子Exon/intron	非同义替换率 Nonsynonymous substitution rate (Ka)	同义替换率 Synonymous substitution rate (Ks)	Ka/Ks	进化时间 Divergent time (Mya)
AT2G30070	AtHAK1	I	712	2,139	3,796	9/8
BnaA03g13690D	BnaA3.HAK1	I	711	2,136	5,533	9/8	0.033	0.489	0.068	16.311
BnaA05g12300D	BnaA5.HAK1	I	714	2,145	3,338	8/7	0.033	0.577	0.057	19.223
BnaC03g16580D	BnaC3.HAK1	I	710	2,133	3,984	9/8	0.034	0.500	0.067	16.676
BnaC04g14750D	BnaC4.HAK1	I	712	2,139	4,392	8/7	0.036	0.494	0.073	16.482
AT2G40540	AtHAK2	J	794	2,385	3,968	9/8
BnaA04g23330D	BnaA4.HAK2	J	776	2,331	3,847	9/8	0.037	0.649	0.056	21.621
BnaC04g01430D	BnaC4.HAK2a	J	784	2,355	6,298	18/17	0.041	0.588	0.070	19.610
BnaC04g47240D	BnaC4.HAK2b	J	777	2,334	3,898	9/8	0.038	0.684	0.055	22.815
AT3G02050	AtHAK3	F	775	2,328	4,453	9/8
BnaA01g13320D	BnaA1.HAK3	F	782	2,349	3,779	8/7	0.015	0.367	0.042	12.228
BnaA09g21950D	BnaA9.HAK3	F	489	1,470	2,821	12/11	0.091	0.674	0.135	22.479
BnaC01g15360D	BnaC1.HAK3	F	705	2,118	3,625	8/7	0.017	0.369	0.045	12.309
AT4G23640	AtHAK4	U	775	2,328	4,453	8/7
BnaC09g24170D	BnaC9.HAK4	U	539	1,620	5,134	10/9	0.080	0.726	0.110	24.201
BnaCnng05490D	BnaCn.HAK4	U	770	2,313	3,546	10/9	0.366
AT4G13420	AtHAK5	T	785	2,358	5,470	9/8
BnaA07g16500D	BnaA7.HAK5	T	747	2,244	7,046	11/10	0.047	0.449	0.104	14.964
BnaC06g15440D	BnaC6.HAK5	T	784	2,355	8,981	9/8	0.050	0.461	0.108	15.383
AT1G70300	AtHAK6	E	782	2,349	3,888	6/5
BnaA02g14820D	BnaA2.HAK6	E	665	1,998	3,831	7/6	0.057	0.699	0.082	23.289
BnaA07g38760D	BnaA7.HAK6	E	700	2,103	3,392	7/6	0.044	0.724	0.060	24.122
BnaC02g19780D	BnaC2.HAK6	E	777	2,334	4,163	6/5	0.052	0.717	0.072	23.893
BnaC06g31400D	BnaC6.HAK6	E	785	2,358	3,467	6/5	0.056	0.794	0.071	26.480
AT5G09400	AtHAK7	R	858	2,577	4,673	10/9
BnaA03g02700D	BnaA3.HAK7	R	864	2,595	3,806	10/9	0.042	0.423	0.099	14.112

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续附表 2　Table S2 continued
基因ID Gene ID	基因名称 Gene name	区块 Block	蛋白长度 Protein length (aa)	编码区长度 Coding sequences length (bp)	DNA全长 DNA length	外显子/内含子Exon/intron	非同义替换率 Nonsynonymous substitution rate (Ka)	同义替换率 Synonymous substitution rate (Ks)	Ka/Ks	进化时间 Divergent time (Mya)
BnaC03g03790D	BnaC3.HAK7	R	864	2,595	3,755	10/9	0.040	0.438	0.092	14.605
AT5G14880	AtHAK8	R	781	2,346	3,951	8/7
BnaA02g02340D	BnaA2.HAK8	R	710	2,133	8,919	20/19	0.026	0.485	0.055	16.177
BnaC02g05800D	BnaC2.HAK8	R	775	2,328	10,988	19/18	0.035	0.560	0.063	18.664
AT4G19960	AtHAK9	U	823	2,472	3,523	9/8
BnaCnng46720D	BnaCn.HAK9	U	794	2,385	4,156	9/8	0.122	0.742	0.164	24.724
BnaA01g10310D	BnaA1.HAK9	U	812	2,439	3,238	8/7	0.053	0.508	0.104	16.949
BnaA03g44320D	BnaA3.HAK9a	U	808	2,427	3,476	9/8	0.075	0.437	0.171	14.570
BnaA03g44330D	BnaA3.HAK9b	U	810	2,433	3,761	10/9	0.087	0.489	0.179	16.291
BnaA08g30510D	BnaA8.HAK9b	U	808	2,427	3,259	8/7	0.048	0.574	0.084	19.138
BnaC01g41320D	BnaC1.HAK9	U	789	2,370	3,584	9/8	0.054	0.505	0.107	16.835
BnaC03g76940D	BnaC3.HAK9	U	804	2,415	3,188	8/7	0.051	0.593	0.085	19.755
BnaC07g36080D	BnaC7.HAK9a	U	709	2,130	3,221	10/9	0.068	0.498	0.136	16.596
BnaC07g36130D	BnaC7.HAK9b	U	798	2,397	3,462	9/8	0.081	0.449	0.180	14.958
BnaC07g36140D	BnaC7.HAK9c	U	402	1,209	2,867	6/5	0.050	0.559	0.090	18.644
AT1G31120	AtHAK10	B	796	2,391	3,859	7/6
BnaA08g08020D	BnaA8.HAK10	B	757	2,274	2,724	6/5	0.040	0.623	0.064	20.763
BnaC08g09300D	BnaC8.HAK10	B	794	2,385	3,563	5/4	0.043	0.639	0.068	21.299
AT2G35060	AtHAK11	J	793	2,382	3,916	8/7
BnaA05g08850D	BnaA5.HAK11	J	787	2,364	3,752	8/7	0.025	0.609	0.040	20.311
BnaC04g10260D	BnaC4.HAK11	J	728	2,187	3,978	9/8	0.022	0.611	0.035	20.379
AT1G60160	AtHAK12	D	827	2,484	3,514	8/7
BnaA01g22220D	BnaA1.HAK12	D	834	2,505	5,005	10/9	0.029	0.366	0.080	12.193
BnaC01g43090D	BnaC1.HAK12	D	832	2,499	4,041	9/8	0.025	0.400	0.063	13.341
AT4G33530	AtHAK13	U	855	2,568	4,430	10/9
BnaA01g03380D	BnaA1.HAK13	U	856	2,571	3,630	10/9	0.054	0.420	0.129	14.011
BnaC01g04660D	BnaC1.HAK13	U	850	2,553	3,831	10/9	0.053	0.411	0.129	13.713

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图 1 拟南芥和甘蓝型油菜中HAKs家族基因系统发育分析

Figure 1. Phylogenetic analysis of HAKs family genes in Arabidopsis and Brassica napus

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图 2 拟南芥和油菜HAKs家族成员的染色体定位

Figure 2. Chromosomal location of HAKs family members in Arabidopsis and Brassica napus

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图 3 HAKs家族基因的顺式作用调控元件富集分析

Figure 3. Enrichment analysis of cis-acting regulatory elements of HAKs family genes

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图 4 甘蓝型油菜HAKs基因家族中AC亚基因组共线性分析

Figure 4. Collinearity analysis of AC subgenome in HAKs gene families in Brassica napus

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图 5 HAKs家族基因在拟南芥、白菜、甘蓝以及甘蓝型油菜之间的共线性分析

Figure 5. Collinearity analysis of HAKs family genes in Arabidopsis, B. rapa, B. oleracea, and B. napus

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图 6 不同营养胁迫下甘蓝型油菜BnaHAKs家族基因的差异表达谱热图

Figure 6. Heat maps of differential expression of BnaHAKs family genes in Brassica napus under different nutrient stresses

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图 7 不同处理下甘蓝型油菜BnaHAKs基因家族差异共表达网络分析

Figure 7. Network analysis of differential co-expression of BnaHAKs gene family in Brassica napus under different treatments

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图 8 油菜BnaA7.HAK5调控低钾胁迫抗性的功能初步解析

Figure 8. Preliminary functional analysis of the regulation of low K stress resistance by BnaA7.HAK5 in Brassica napus

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附图 1 HAKs家族基因跨膜特性

Supplementary figure 1. Transmembrane characteristics of HAKs family genes

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表 1 拟南芥和芸薹属作物中HAKs家族基因的拷贝数

Table 1 Gene copy numbers of HAKs family in Arabidopsis and Brassica crops

基因名称 Gene name	拟南芥 Arabidopsis thaliana (125 Mb)	白菜 Brassica rapa (465 Mb)	甘蓝 Brassica oleracea (485 Mb)	甘蓝型油菜 Brassica napus
HAK1	1	3	1	4
HAK2	1	1	2	3
HAK3	1	2	2	3
HAK4	1	2	2	2
HAK5	1	1	1	2
HAK6	1	1	2	4
HAK7	1	1	2	2
HAK8	1	1	2	2
HAK9	1	3	4	10
HAK10	1	2	1	2
HAK11	1	2	2	2
HAK12	1	1	1	2
HAK13	1	1	1	2
总计 Total	13	21	23	40

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1 拟南芥和油菜中HAKs家族基因的理化性质

1 Physicochemical properties of HAKs family genes in Arabidopsis and Brassica napus

基因ID Gene ID	基因名称 Gene name	不稳定系数 Instability index	脂肪指数 Aliphatic index	平均亲水系数 Average hydrophobic index	跨膜结构域 Trans-membrane domains	亚细胞定位 Subcellular localization
AT2G30070	AtHAK1	39.39	105.72	0.483	13	Endoplamic reticulum, mitochondrion, nucleus, plasmolemma, vacuole
BnaA03g13690D	BnaA3.HAK1	37.36	107.79	0.485	13	Chloroplast, endoplamic reticulum, plasmolemma
BnaA05g12300D	BnaA5.HAK1	36.15	105.97	0.459	13	Chloroplast, cytoplasm, endoplamic reticulum, plasmolemma
BnaC03g16580D	BnaC3.HAK1	36.68	109.32	0.499	13	Chloroplast, endoplamic reticulum, plasmolemma
BnaC04g14750D	BnaC4.HAK1	35.74	106.81	0.48	12	Endoplamic reticulum, plasmolemma, vacuole
AT2G40540	AtHAK2	45.54	108.12	0.349	12	Chloroplast, endoplamic reticulum, plasmolemma
BnaA04g23330D	BnaA4.HAK2	42.75	108.36	0.349	12	Chloroplast, endoplamic reticulum, plasmolemma, vacuole
BnaC04g01430D	BnaC4.HAK2a	45.39	101.44	0.133	13	Chloroplast, endoplamic reticulum, plasmolemma, vacuole
BnaC04g47240D	BnaC4.HAK2b	42.85	107.84	0.342	12	Chloroplast, endoplamic reticulum, plasmolemma, vacuole
AT3G02050	AtHAK3	43.85	107.16	0.434	14	Endoplamic reticulum, plasmolemma
BnaA01g13320D	BnaA1.HAK3	43.79	106.19	0.427	12	Plasmolemma
BnaA09g21950D	BnaA9.HAK3	44.12	107.45	0.557	11	Endoplamic reticulum, plasmolemma, vacuole
BnaC01g15360D	BnaC1.HAK3	43.31	106.31	0.438	10	Endoplamic reticulum, plasmolemma, vacuole
AT4G23640	AtHAK4	43.85	107.16	0.434	14	Endoplamic reticulum, golgi apparatus, plasmolemma
BnaC09g24170D	BnaC9.HAK4	41.62	111.74	0.642	15	Endoplamic reticulum, golgi apparatus, plasmolemma
BnaCnng05490D	BnaCn.HAK4	44.9	107.83	0.391	13	Plasmolemma
AT4G13420	AtHAK5	29.71	101.38	0.222	12	Endoplamic reticulum, plasmolemma
BnaA07g16500D	BnaA7.HAK5	28.38	100.43	0.194	10	Endoplamic reticulum, plasmolemma
BnaC06g15440D	BnaC6.HAK5	28.48	100.04	0.192	12	Endoplamic reticulum, plasmolemma
AT1G70300	AtHAK6	46.34	105.81	0.298	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaA02g14820D	BnaA2.HAK6	47.18	111.08	0.445	12	Plasmolemma, vacuole
BnaA07g38760D	BnaA7.HAK6	42.73	110.1	0.414	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaC02g19780D	BnaC2.HAK6	45.08	106.85	0.325	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaC06g31400D	BnaC6.HAK6	45.3	104.37	0.251	12	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
AT5G09400	AtHAK7	41.87	105.59	0.297	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaA03g02700D	BnaA3.HAK7	43.61	104.31	0.268	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole

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续附表 1　Table S1 continued
基因ID Gene ID	基因名称 Gene name	不稳定系数 Instability index	脂肪指数 Aliphatic index	平均亲水系数 Average hydrophobic index	跨膜结构域 Trans-membrane domains	亚细胞定位 Subcellular localization
BnaC03g03790D	BnaC3.HAK7	43.03	104.76	0.271	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
AT5G14880	AtHAK8	38.5	107.41	0.306	13	Endoplamic reticulum, mitochondrion, plasmolemma, vacuole
BnaA02g02340D	BnaA2.HAK8	43.03	87.1	-0.145	13	Endoplamic reticulum, mitochondrion, plasmolemma
BnaC02g05800D	BnaC2.HAK8	42.98	87.03	-0.137	15	Endoplamic reticulum, mitochondrion, nucleus, plasmolemma
AT4G19960	AtHAK9	34.64	105.01	0.298	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA01g10310D	BnaA1.HAK9	35.67	105.7	0.287	13	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA03g44320D	BnaA3.HAK9a	33.96	106.1	0.259	13	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA03g44330D	BnaA3.HAK9b	32.73	106.58	0.273	13	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaA08g30510D	BnaA8.HAK9b	38.04	107.57	0.278	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaC01g41320D	BnaC1.HAK9	36.57	106.92	0.342	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaC03g76940D	BnaC3.HAK9	37.14	107.5	0.296	14	Endoplamic reticulum, golgi apparatus plasmolemma, vacuole
BnaC07g36080D	BnaC7.HAK9a	38.62	105.78	0.23	11	Cytoplasm, endoplamic reticulum, nucleus, plasmolemma, vacuole
BnaC07g36130D	BnaC7.HAK9b	35.13	105.73	0.284	13	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaC07g36140D	BnaC7.HAK9c	29.93	114.98	0.504	9	Chloroplast, cytoplasm, endoplamic reticulum, plasmolemma, vacuole
BnaCnng46720D	BnaCn.HAK9	30.02	107.85	0.318	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
AT1G31120	AtHAK10	40.19	103.54	0.3	11	Endoplamic reticulum, plasmolemma
BnaA08g08020D	BnaA8.HAK10	40.07	106.43	0.341	11	Endoplamic reticulum, plasmolemma
BnaC08g09300D	BnaC8.HAK10	37.98	103.44	0.289	11	Endoplamic reticulum, plasmolemma
AT2G35060	AtHAK11	37.2	105.79	0.322	11	Endoplamic reticulum, plasmolemma, vacuole
BnaA05g08850D	BnaA5.HAK11	38.04	106.1	0.35	13	Endoplamic reticulum, plasmolemma, vacuole
BnaC04g10260D	BnaC4.HAK11	38.26	109.62	0.412	14	Endoplamic reticulum, plasmolemma, vacuole
AT1G60160	AtHAK12	42.72	108.33	0.376	13	Endoplamic reticulum, plasmolemma
BnaA01g22220D	BnaA1.HAK12	42.3	108.28	0.395	13	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaC01g43090D	BnaC1.HAK12	40.31	108.73	0.395	13	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
AT4G33530	AtHAK13	39.94	109.43	0.331	11	Endoplamic reticulum, golgi apparatus, mitochondrion, plasmolemma, vacuole
BnaA01g03380D	BnaA1.HAK13	40.87	108.07	0.329	12	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole
BnaC01g04660D	BnaC1.HAK13	42.84	108.72	0.333	11	Endoplamic reticulum, golgi apparatus, plasmolemma, vacuole

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2 拟南芥和油菜中HAKs家族基因的分子特征

2 Molecular characteristics of HAKs family genes in Arabidopsis and Brassica napus

基因ID Gene ID	基因名称 Gene name	区块 Block	蛋白长度 Protein length (aa)	编码区长度 Coding sequences length (bp)	DNA全长 DNA length	外显子/内含子Exon/intron	非同义替换率 Nonsynonymous substitution rate (Ka)	同义替换率 Synonymous substitution rate (Ks)	Ka/Ks	进化时间 Divergent time (Mya)
AT2G30070	AtHAK1	I	712	2,139	3,796	9/8
BnaA03g13690D	BnaA3.HAK1	I	711	2,136	5,533	9/8	0.033	0.489	0.068	16.311
BnaA05g12300D	BnaA5.HAK1	I	714	2,145	3,338	8/7	0.033	0.577	0.057	19.223
BnaC03g16580D	BnaC3.HAK1	I	710	2,133	3,984	9/8	0.034	0.500	0.067	16.676
BnaC04g14750D	BnaC4.HAK1	I	712	2,139	4,392	8/7	0.036	0.494	0.073	16.482
AT2G40540	AtHAK2	J	794	2,385	3,968	9/8
BnaA04g23330D	BnaA4.HAK2	J	776	2,331	3,847	9/8	0.037	0.649	0.056	21.621
BnaC04g01430D	BnaC4.HAK2a	J	784	2,355	6,298	18/17	0.041	0.588	0.070	19.610
BnaC04g47240D	BnaC4.HAK2b	J	777	2,334	3,898	9/8	0.038	0.684	0.055	22.815
AT3G02050	AtHAK3	F	775	2,328	4,453	9/8
BnaA01g13320D	BnaA1.HAK3	F	782	2,349	3,779	8/7	0.015	0.367	0.042	12.228
BnaA09g21950D	BnaA9.HAK3	F	489	1,470	2,821	12/11	0.091	0.674	0.135	22.479
BnaC01g15360D	BnaC1.HAK3	F	705	2,118	3,625	8/7	0.017	0.369	0.045	12.309
AT4G23640	AtHAK4	U	775	2,328	4,453	8/7
BnaC09g24170D	BnaC9.HAK4	U	539	1,620	5,134	10/9	0.080	0.726	0.110	24.201
BnaCnng05490D	BnaCn.HAK4	U	770	2,313	3,546	10/9	0.366
AT4G13420	AtHAK5	T	785	2,358	5,470	9/8
BnaA07g16500D	BnaA7.HAK5	T	747	2,244	7,046	11/10	0.047	0.449	0.104	14.964
BnaC06g15440D	BnaC6.HAK5	T	784	2,355	8,981	9/8	0.050	0.461	0.108	15.383
AT1G70300	AtHAK6	E	782	2,349	3,888	6/5
BnaA02g14820D	BnaA2.HAK6	E	665	1,998	3,831	7/6	0.057	0.699	0.082	23.289
BnaA07g38760D	BnaA7.HAK6	E	700	2,103	3,392	7/6	0.044	0.724	0.060	24.122
BnaC02g19780D	BnaC2.HAK6	E	777	2,334	4,163	6/5	0.052	0.717	0.072	23.893
BnaC06g31400D	BnaC6.HAK6	E	785	2,358	3,467	6/5	0.056	0.794	0.071	26.480
AT5G09400	AtHAK7	R	858	2,577	4,673	10/9
BnaA03g02700D	BnaA3.HAK7	R	864	2,595	3,806	10/9	0.042	0.423	0.099	14.112

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续附表 2　Table S2 continued
基因ID Gene ID	基因名称 Gene name	区块 Block	蛋白长度 Protein length (aa)	编码区长度 Coding sequences length (bp)	DNA全长 DNA length	外显子/内含子Exon/intron	非同义替换率 Nonsynonymous substitution rate (Ka)	同义替换率 Synonymous substitution rate (Ks)	Ka/Ks	进化时间 Divergent time (Mya)
BnaC03g03790D	BnaC3.HAK7	R	864	2,595	3,755	10/9	0.040	0.438	0.092	14.605
AT5G14880	AtHAK8	R	781	2,346	3,951	8/7
BnaA02g02340D	BnaA2.HAK8	R	710	2,133	8,919	20/19	0.026	0.485	0.055	16.177
BnaC02g05800D	BnaC2.HAK8	R	775	2,328	10,988	19/18	0.035	0.560	0.063	18.664
AT4G19960	AtHAK9	U	823	2,472	3,523	9/8
BnaCnng46720D	BnaCn.HAK9	U	794	2,385	4,156	9/8	0.122	0.742	0.164	24.724
BnaA01g10310D	BnaA1.HAK9	U	812	2,439	3,238	8/7	0.053	0.508	0.104	16.949
BnaA03g44320D	BnaA3.HAK9a	U	808	2,427	3,476	9/8	0.075	0.437	0.171	14.570
BnaA03g44330D	BnaA3.HAK9b	U	810	2,433	3,761	10/9	0.087	0.489	0.179	16.291
BnaA08g30510D	BnaA8.HAK9b	U	808	2,427	3,259	8/7	0.048	0.574	0.084	19.138
BnaC01g41320D	BnaC1.HAK9	U	789	2,370	3,584	9/8	0.054	0.505	0.107	16.835
BnaC03g76940D	BnaC3.HAK9	U	804	2,415	3,188	8/7	0.051	0.593	0.085	19.755
BnaC07g36080D	BnaC7.HAK9a	U	709	2,130	3,221	10/9	0.068	0.498	0.136	16.596
BnaC07g36130D	BnaC7.HAK9b	U	798	2,397	3,462	9/8	0.081	0.449	0.180	14.958
BnaC07g36140D	BnaC7.HAK9c	U	402	1,209	2,867	6/5	0.050	0.559	0.090	18.644
AT1G31120	AtHAK10	B	796	2,391	3,859	7/6
BnaA08g08020D	BnaA8.HAK10	B	757	2,274	2,724	6/5	0.040	0.623	0.064	20.763
BnaC08g09300D	BnaC8.HAK10	B	794	2,385	3,563	5/4	0.043	0.639	0.068	21.299
AT2G35060	AtHAK11	J	793	2,382	3,916	8/7
BnaA05g08850D	BnaA5.HAK11	J	787	2,364	3,752	8/7	0.025	0.609	0.040	20.311
BnaC04g10260D	BnaC4.HAK11	J	728	2,187	3,978	9/8	0.022	0.611	0.035	20.379
AT1G60160	AtHAK12	D	827	2,484	3,514	8/7
BnaA01g22220D	BnaA1.HAK12	D	834	2,505	5,005	10/9	0.029	0.366	0.080	12.193
BnaC01g43090D	BnaC1.HAK12	D	832	2,499	4,041	9/8	0.025	0.400	0.063	13.341
AT4G33530	AtHAK13	U	855	2,568	4,430	10/9
BnaA01g03380D	BnaA1.HAK13	U	856	2,571	3,630	10/9	0.054	0.420	0.129	14.011
BnaC01g04660D	BnaC1.HAK13	U	850	2,553	3,831	10/9	0.053	0.411	0.129	13.713

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1. 材料与方法
1.1 HAKs家族基因序列的检索及命名
1.2 HAKs家族基因的理化性质分析及染色体定位
1.3 HAKs家族基因的共线性分析及选择压力分析
1.4 HAKs家族基因的多序列比对和系统发育分析
1.5 HAKs家族基因的保守基序/域和基因结构分析
1.6 HAKs家族基因的启动子调控元件分析
1.7 植物材料培养及处理
1.8 不同营养胁迫下BnaHAKs家族基因的转录及基因共表达网络分析
1.9 低钾条件下实时荧光定量PCR (RT-qPCR)分析
1.10 亚细胞定位分析
1.11 矿质元素含量的测定
2. 结果与分析
2.1 HAKs家族基因的全基因组鉴定
2.2 HAKs家族基因的分子特征
2.3 HAKs家族基因的系统发育分析
2.4 HAKs家族基因的染色体定位
2.5 HAKs家族基因的顺式作用调控元件分析
2.6 HAKs家族基因共线性分析
2.7 不同养分胁迫下BnaHAKs的转录分析
2.8 油菜BnaA7.HAK5调控低钾胁迫抗性的功能初步解析
3. 讨论
3.1 综合生物信息学分析全面揭示了BnaHAKs的分子特征
3.2 BnaHAKs家族成员在响应不同逆境胁迫中具有复杂性
3.3 BnaA7.HAK5可能在油菜响应低钾胁迫抗性反应中发挥核心作用
4. 结论

1. 材料与方法
1.1 HAKs家族基因序列的检索及命名
1.2 HAKs家族基因的理化性质分析及染色体定位
1.3 HAKs家族基因的共线性分析及选择压力分析
1.4 HAKs家族基因的多序列比对和系统发育分析
1.5 HAKs家族基因的保守基序/域和基因结构分析
1.6 HAKs家族基因的启动子调控元件分析
1.7 植物材料培养及处理
1.8 不同营养胁迫下BnaHAKs家族基因的转录及基因共表达网络分析
1.9 低钾条件下实时荧光定量PCR (RT-qPCR)分析
1.10 亚细胞定位分析
1.11 矿质元素含量的测定
2. 结果与分析
2.1 HAKs家族基因的全基因组鉴定
2.2 HAKs家族基因的分子特征
2.3 HAKs家族基因的系统发育分析
2.4 HAKs家族基因的染色体定位
2.5 HAKs家族基因的顺式作用调控元件分析
2.6 HAKs家族基因共线性分析
2.7 不同养分胁迫下BnaHAKs的转录分析
2.8 油菜BnaA7.HAK5调控低钾胁迫抗性的功能初步解析
3. 讨论
3.1 综合生物信息学分析全面揭示了BnaHAKs的分子特征
3.2 BnaHAKs家族成员在响应不同逆境胁迫中具有复杂性
3.3 BnaA7.HAK5可能在油菜响应低钾胁迫抗性反应中发挥核心作用
4. 结论

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异源四倍体油菜HAKs家族核心基因的鉴定及其功能初步解析

作者简介: 陈俊帆 E-mail: hyp19890413@163.com

通讯作者: 黄进勇 E-mail: jinyhuang@zzu.edu.cn 华营鹏 E-mail: yingpenghua@zzu.edu.cn

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