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作物学报 ›› 2023, Vol. 49 ›› Issue (7): 1871-1881.doi: 10.3724/SP.J.1006.2023.24134

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于干旱胁迫转录组信息的蚕豆ASPAT基因家族分析

魏正欣1(), 刘昌燕2(), 陈宏伟2, 李莉2, 孙龙清2, 韩雪松2, 焦春海2,*(), 沙爱华1,*()   

  1. 1长江大学农学院 / 湿地生态与农业利用教育部工程研究中心, 湖北荆州 434025
    2湖北省农业科学院粮食作物研究所 / 粮食作物种质创新与遗传改良湖北省重点实验室, 湖北武汉 430064
  • 收稿日期:2022-06-03 接受日期:2022-11-25 出版日期:2023-07-12 网络出版日期:2022-12-01
  • 通讯作者: *沙爱华, E-mail: aihuasha@163.com; 焦春海, E-mail: jiaoch@hotmai.com
  • 作者简介:魏正欣, E-mail: 2545439813@qq.com
    刘昌燕, E-mail: Liucy0602@163.com第一联系人：**同等贡献
  • 基金资助:
    本研究由国家重点研发计划项目(2019YFD1001303);本研究由国家重点研发计划项目(2019YFD1001300);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-08);湖北省农业科技创新中心项目(2021-620-000-001-01)

Analysis of ASPAT gene family based on drought-stressed transcriptome sequencing in Vicia faba L.

WEI Zheng-Xin1(), LIU Chang-Yan2(), CHEN Hong-Wei2, LI Li2, SUN Long-Qing2, HAN Xue-Song2, JIAO Chun-Hai2,*(), SHA Ai-Hua1,*()   

  1. 1College of Agriculture, Yangtze University / Engineering Research center of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434025, Hubei, China
    2Hubei Academy of Agricultural Sciences Institute of Food Crops / Hubei Provincial Key Laboratory of Germplasm Innovation and Genetic Improvement of Food Crops, Wuhan 430064, Hubei, China
  • Received:2022-06-03 Accepted:2022-11-25 Published:2023-07-12 Published online:2022-12-01
  • Contact: *E-mail: aihuasha@163.com; E-mail: jiaoch@hotmai.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    The National Key Research and Development Program of China(2019YFD1001303);The National Key Research and Development Program of China(2019YFD1001300);The China Agriculture Research System of MOF and MARA(CARS-08);The Hubei Agricultural Science and Technology Innovation Center Project(2021-620-000-001-01)

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摘要：

蚕豆(Vicia faba L.)是一种重要的食用豆作物, 干旱会导致蚕豆产量降低。鉴定蚕豆耐旱基因, 有利于通过分子标记辅助育种和基因工程手段培育抗旱品种。在前期研究中, 我们通过转录组测序鉴定出了一类天冬氨酸转氨酶基因(ASPAT)响应蚕豆干旱胁迫。ASPAT催化天冬氨酸(ASP)和α-酮戊二酸的可逆转氨反应, 生成草酰乙酸和谷氨酸, 是参与植物体代谢的关键酶。本文基于转录组测序数据, 通过生物信息学方法全基因组鉴定了蚕豆ASPAT基因家族成员, 并分析了各基因家族成员的理化性质、亚细胞定位、基因结构、蛋白结构域、保守基序、系统进化、蛋白质互作和基因表达模式。结果显示, 蚕豆转录参考基因组中共有8个ASPAT基因家族成员, 包括3个真核型ASPAT (AAT)基因和5个原核型ASPAT (PAT)基因, 分别定位于线粒体和叶绿体。系统发育分析表明, 蚕豆ASPAT蛋白主要可分为2个亚族Iα和Iβ。Iα为AAT蛋白, 包括VfASPAT1~VfASPAT3。Iβ为PAT蛋白, 包括VfASPAT4~VfASPAT8。蚕豆ASPAT家族成员均含有motif 1、motif 3、motif 9, 且具有相似的基因结构和共同的蛋白结构域Aminotran-1-2。VfASPAT蛋白互作网络分析表明该家族成员可能参与逆境胁迫代谢过程。转录组和荧光定量PCR分析表明该家族8个成员在干旱处理16 h和64 h表达模式不同, 除VfASPAT4和VfASPAT6外其他基因表达量都呈上调趋势, 表明VfASPAT基因可能对干旱胁迫有正向调控作用。本文研究结果为进一步分析蚕豆ASPAT的抗旱功能, 应用ASPAT提高蚕豆抗旱性奠定了基础。

关键词: 蚕豆, 天冬氨酸转氨酶, 基因家族, 干旱胁迫, 基因表达

Abstract:

Fava beans (Vicia faba L.) is an important food bean crop, and drought can lead to lower fava bean yields. The identification of drought tolerance genes of fava beans is beneficial to breed the drought-resistant varieties through molecular marker-assisted breeding and genetic engineering. In a previous study, we identified a class of aspartate aminotransferase genes (ASPAT) in response to faba bean drought stress by transcriptome sequencing. ASPAT catalyzes the reversible reaction of transamination between aspartate and α-oxoglutarate to generate oxaloacetate and glutamate, and plays a key role in plant metabolism. Based on transcriptomic sequencing data, ASPAT members were identified in genome-wide level. The physicochemical properties, subcellular localizations, gene structures, protein domains, conserved motifs, phylogeny, protein interaction, and gene expression patterns of each member were analyzed. The results indicate that eight ASPAT genes were identified from the transcriptomes, which included three eukaryotype ASPAT (AAT) genes and five prokarytoype ASPAT (PAT) genes with mitochondria and chloroplast location, respectively. Phylogenetic analysis showed that ASPAT could be divided into two subfamilies Iα and Iβ. Iα is AAT protein including VfASPAT1-VfASPAT3, and Iβ is PAT protein consisting of VfASPAT4-VfASPAT8. ASPAT family members of Vicia faba contain motif 1, motif 3, and motif 9, and had similar gene structures and common protein domain aminotran-1-2. VfASPATs protein interaction network suggested that the members might be involved in stress metabolism. Transcriptome and qRT-PCR indicated that he expression patterns of the eight members were different at 16 hours and 64 hours under drought stress. All the members were up-regulated with the exception of VfASPAT4 and VfASPAT6, implying that VfASPATs may have a positive regulation effect on drought stress. In this study, eight ASPAT genes of Vicia faba were identified, and the structures, distribution, evolutionary relationship, and the relative expression patterns of ASPAT gene family members were analyzed. The results of this study laid a foundation for further analysis of drought resistance function of VfASPATs, and the application of ASPAT to improve drought resistance of fava bean.

Key words: Vicia faba L., aspartate aminotransferase (ASPAT), gene family, drought stress, gene expression

表1

实时荧光定量PCR所用的引物"

基因Gene name 正向引物序列Forward sequence (5'-3') 反向引物序列 Reverse sequence (5'-3')
VfASPAT1 AAGGATAAACGAATAGCCGCAGT TCGGGATGATAGTAACGGAATGT
VfASPAT2 GGCTTCTCACATTTCTCCTTCTC CAACTCGCCTAACTACATCCAAC
VfASPAT3 TGAAGAATTGCAGCCGTATG TGGCAACTCTTTGCTGTTTG
VfASPAT4 ATTGAGGTGGAGCAGAACAGA CGCCAAGCGAATAACAGGAAC
VfASPAT5 GGTGATGAGGTTATTGTGTTTGC CTTTCCAGTAGGATTGTGAGGG
VfASPAT6 AAGCCCTCGCAGATCGGAACAC CCAAAAACTCCCATAGGCACAA
VfASPAT7 AAAATGGAAGGTAAACTGTCGC CCTGTACTGATTGAGGTCGGAA
VfASPAT8 CTGCAGCAACAAGGGAACAAC TGGCAACTCTTTGCTGTTTG
VfNADHD4 AGGGTTAGTGAGCACCATGC ATAGCCAAAGGGAATACGCC

表2

干旱胁迫处理两蚕豆品种发芽率比较"

处理时间
Processing time		 鄂蚕豆1号-T
Ecandou 1-T		 CDAS105-T 鄂蚕豆1号-CK
Ecandou 1-CK		 CDAS105-CK
48 h 0 0 47.6 cd 61.9 bc
64 h 0 4.8 e 78.6 a 76.2 a
72 h 0 9.5 e 83.3 a 81.0 a
96 h 9.5 e 38.1 d 88.1 a 85.7 a

表3

蚕豆ASPAT蛋白理化性质及亚细胞定位预测"

蛋白质
Protein		 氨基酸数目Number of amino acids 等电点
pI		 相对分子质量
Molecular weight		 脂融指数Aliphatic index 不稳定指数Instability index 疏水性分值GARVY 亚细胞定位
Subcellular
localization
VfASPAT1 424 8.42 46,958.64 82.38 39.68 -0.261 线粒体Mitochondrion
VfASPAT2 417 7.24 45,448.99 96.91 43.76 -0.052 叶绿体Chloroplast
VfASPAT3 453 7.73 49,658.54 84.86 36.02 -0.197 叶绿体, 线粒体
Chloroplast, mitochondrion
VfASPAT4 481 7.60 52,060.70 96.36 36.72 0.039 叶绿体Chloroplast
VfASPAT5 394 6.22 43,469.05 85.91 29.62 -0.063 叶绿体Chloroplast
VfASPAT6 407 5.59 45,024.06 96.61 35.48 0.030 叶绿体Chloroplast
VfASPAT7 409 6.02 44,829.10 89.51 41.14 -0.077 叶绿体Chloroplast
VfASPAT8 459 6.56 49,923.62 79.48 50.90 -0.147 叶绿体Chloroplast

表4

蚕豆ASPAT蛋白二级结构预测分析"

蛋白质
Protein		 α-螺旋
Alpha helix		 β-转角
Beta turn		 延伸链
Extended strand		 无规则卷曲
Random coil
VfASPAT1 45.28 7.55 14.39 32.78
VfASPAT2 43.65 6.95 15.11 34.29
VfASPAT3 40.84 7.06 17.44 34.66
VfASPAT4 39.09 7.07 18.50 35.34
VfASPAT5 42.39 8.63 17.51 31.47
VfASPAT6 43.49 7.13 14.99 34.40
VfASPAT7 41.08 7.33 19.07 32.52
VfASPAT8 35.29 8.28 20.04 36.38

图1

蚕豆ASPAT蛋白家族的进化分析"

图2

VfASPAT基因结构、蛋白结构域和保守基序分析 A: 基因结构; B: 蛋白结构域; C: motif; D: motif氨基酸序列。"

图3

蚕豆和拟南芥ASPAT蛋白磷酸吡哆醛结合位点 红色矩形框分别为AAT和PAT蛋白磷酸吡哆醛结合位点。"

图4

蚕豆ASPAT蛋白质互作网络"

图5

转录组鉴定蚕豆ASPAT表达 CK1-16/CK1-64为鄂蚕豆1号正常处理16 h/64 h; CK2-16/CK2-64为CDAS105正常处理16 h/64 h; T1-16/T1-64为鄂蚕豆1号干旱处理16 h/64 h; T2-16/T2-64为CDAS105干旱处理16 h/64 h。"

图6

实时荧光定量PCR分析VfASPAT在鄂蚕豆1号和CDAS105中的表达差异 CK-16和CK-64分别为正常处理16 h和64 h时CDAS105/鄂蚕豆1号的表达量比值; T1-16和T1-64分别为干旱处理16 h和64 h时CDAS105/鄂蚕豆1号的表达量比值。小写字母表示差异显著性(P < 0.05)。"

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