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作物杂志,2018, 第4期: 53–61 doi: 10.16035/j.issn.1001-7283.2018.04.010

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

甘蔗ScHAK10基因克隆及表达分析

罗海斌1,蒋胜理1,黄诚梅1,曹辉庆1,邓智年2,吴凯朝2,徐林2,陆珍1,魏源文1   

  1. 1 广西作物遗传改良生物技术重点开放实验室,530007,广西南宁
    2 广西农业科学院甘蔗研究所/农业农村部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良重点实验室,530007,广西南宁
  • 收稿日期:2018-01-25 修回日期:2018-06-15 出版日期:2018-08-15 发布日期:2018-08-23
  • 作者简介:作者简介:罗海斌,助理研究员,从事甘蔗分子育种研究
  • 基金资助:
    广西自然科学基金(2014GXNSFAA118081;2013GXNSFAA019077);广西八桂学者专项项目([2013]3号);广西农业科学院基本科研业务专项项目(2015YT96;桂农科2017YM35)

Cloning and Expression of ScHAK10 Gene in Sugarcane

Luo Haibin1,Jiang Shengli1,Huang Chengmei1,Cao Huiqing1,Deng Zhinian2,Wu Kaichao2,Xu Lin2,Lu Zhen1,Wei Yuanwen1   

  1. 1 Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Nanning 530007, Guangxi, China
    2 Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences/Key Laboratory of Sugarcane Biotechnology and Genetic Improvement of Guangxi, Ministry of Agriculture and Rural Affairs, Nanning 530007, Guangxi, China
  • Received:2018-01-25 Revised:2018-06-15 Online:2018-08-15 Published:2018-08-23

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摘要：

为探究甘蔗HAK基因家族中ScHAK10基因的功能及干旱环境下的响应机制,利用同源克隆技术（homologous cloning）从新台糖22号中克隆得到ScHAK10基因的完整编码序列,并对其进行生物信息学分析,同时采用q-PCR技术分析基因在不同组织以及在不同干旱阶段的表达情况。结果表明,ScHAK10基因编码区全长为2 433bp,编码810个氨基酸,相对分子量89.83kD,等电点（pI）为8.46,预测其为疏水碱性蛋白;核苷酸同源性分析表明ScHAK10基因与玉米（Zea mays L.）、高粱[Sorghum bicolor （L.） Moench]等其他作物中HAK基因具有高度的同源性;该蛋白具有跨膜结构域,定位在细胞质膜上。蛋白质二级结构分析发现其主要由α螺旋（38.15%）、扩展长链（19.26%）和无规则卷曲（37.16%）组成;蛋白功能预测显示其与阳离子运输通道相关,有较大的概率显示其介入转录、信号传导、免疫应答等生物活动。qPCR表达分析显示,ScHAK10基因在不同部位都有表达,叶片中的表达量最高,其次为茎,根系中的表达量最低。干旱胁迫下ScHAK10基因受到诱导表达,表达水平根据胁迫程度的加深而呈现出先上调后下降的表达趋势,复水后,基因表达量降低。本研究为进一步阐明甘蔗ScHAK10基因的功能及甘蔗耐旱调控相关分子机制奠定了基础。

关键词: 甘蔗, 钾转运蛋白基因（HAK）, 基因克隆, 生物信息学分析, 基因表达

Abstract:

To explore the function of ScHAK10 in Sugarcane HAK gene family and molecular response mechanism under drought stress, the gene ScHAK10 coding sequence (CDS) from the ROC22 was cloned by homologous cloning. Sequencing and bioinformatics analysis indicated that ScHAK10 sequence obtained was 2 433bp in length, coding 810 amino acids, the molecular weight of ScHAK10 was 89.83kD, isoelectric point (pI) was 8.46, hydrophobic basic protein was prediction. Sequence comparison with maize (Zea mays L.), sorghum [Sorghum bicolor (L.) Moench] and other crops showed that ScHAK10 gene was highly homologous with that of other species. ScHAK10 protein contains multiple trans-membrane domains so that it is predicted to be located on the cytoplasmic membrane. Secondary structure analysis showed that it had alpha helix (38.15%), extended long chain (19.26%) and irregular curling (37.16%). Prediction of protein function showed that it may be involved in biological reactions such as transcription, signal transduction, and immune responses. Real-time PCR result indicated that ScHAK10 expressed in different tissues, the highest expression in leaf, followed by stem, and the lowest in root. ScHAK10 gene expression was up-regulated by drought stress. According to the deepening of the stress level, the expression of ScHAK10 rose initially, and then dropped, and decreased after rewaterring. These results indicated that contributes were made for further illustrating its function and its molecular mechanism in the regulation of sugarcane drought.

Key words: Sugarcane, Potassium transporter gene (HAK), Gene cloning, Bioinformatics analysis, Gene expression

图1

ScHAK10基因的RT-PCR扩增电泳结果"

图2

ScHAK10基因编码区的核酸序列及其推导氨基酸序列"

表1

ScHAK10与其他植物HAK基因的核苷酸同源性比对分析结果"

登录号Accession No. 物种Species 同源性Homology (%) 基因Gene
XM_021449505.1 高粱 95 potassium transporter 10
KF815907.1 玉米 94 high-affinity potassium transporter10
XM_004965768.3 小米 90 potassium transporter 10
XM_015788218.1 水稻 87 potassium transporter 10
XM_020332943.1 山羊草 86 potassium transporter 10
XM_003563544.3 二穗短柄草 85 potassium transporter 10
XM_010943891.2 油棕 80 potassium transporter 10

图3

ScHAK10蛋白的亲疏水性预检测结果|||横坐标表示氨基酸位置;疏水和亲水位点分别在零水平线的上方和下方"

图4

利用TMHMM分析ScHAK10蛋白跨膜区域"

图5

利用SignalP 4.1 Server 分析ScHAK10信号肽"

图6

ScHAK10蛋白的二级结构预测结果"

图7

ScHAK10蛋白的三级结构预测结果"

表2

ScHAK10蛋白功能分类结果"

功能分类Functional category 概率Probability 比值
Odds		 基因本体分类
Gene ontology category		 概率
Probability		 比值
Odds
运输和结合Transport and binding 0.773 1.885 电压门控性离子通道Voltage-gated ion channel 0.279 12.682
嘌呤和嘧啶Purines and pyrimidines 0.331 1.362 转录Transcription 0.219 1.711
辅酶合成Biosynthesis of cofactors 0.210 2.917 信号传导Signal transducer 0.205 0.958
脂肪酸代谢Fatty acid metabolism 0.017 1.308 离子通道Ion channel 0.169 2.965
运输Translation 0.071 1.614 阳离子通道Cation channel 0.146 3.174

图8

基于HAK基因构建的系统发育进化树 系统发育树采用邻接法,置信度采用500次重复计算,分子节点的数值代表置信度,标尺代表每个核苷酸位置发生0.05次替代"

图9

ScHAK10与其他植物HAK蛋白的序列比对"

图10

甘蔗ScHAK10基因在不同组织中的表达 误差线为每组处理的标准误差（n=3）。下同"

图11

ScHAK10基因在干旱肋迫下表达变化"

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