生物技术通报 ›› 2023, Vol. 39 ›› Issue (2): 107-115.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0568
庞强强1,2,3(), 孙晓东1,2,3(), 周曼1,2,3, 蔡兴来1,2,3, 张文1,2,3, 王亚强1,2,3
收稿日期:
2022-05-09
出版日期:
2023-02-26
发布日期:
2023-03-07
作者简介:
庞强强,男,硕士,助理研究员,研究方向:菜心栽培与育种;E-mail: 基金资助:
PANG Qiang-qiang1,2,3(), SUN Xiao-dong1,2,3(), ZHOU Man1,2,3, CAI Xing-lai1,2,3, ZHANG Wen1,2,3, WANG Ya-qiang1,2,3
Received:
2022-05-09
Published:
2023-02-26
Online:
2023-03-07
摘要:
为探索菜心对高温胁迫的响应机制,发掘耐热相关基因,本研究采用全基因合成法(PAS)对前期从菜心高温胁迫转录组数据库中筛选到的1个差异表达基因进行克隆,并开展相关生物信息学分析,利用实时荧光定量PCR技术(RT-qPCR)分析该基因在高温胁迫下的表达模式。结果表明,克隆得到开放阅读框(ORF)为906 bp的基因,命名为BrHsfA3。该基因编码301个氨基酸,蛋白相对分子量为34.12 kD,理论等电点为5.00,为无信号肽的亲水性蛋白,存在丝氨酸、苏氨酸和酪氨酸3个磷酸化位点。BrHsfA3蛋白含有1个Hsf保守结构域,具有典型的DNA结合结构域(DBD)、寡聚化结构域(OD)、核定位信号结构域(NLS)和转录激活结构域(AHA),蛋白二级结构以α-螺旋和无规则卷曲为主。进化分析发现,BrHsfA3基因与甘蓝型油菜的亲缘关系最近。RT-qPCR分析结果显示,BrHsfA3在耐热自交系CX1-7叶、花中受高温诱导后表达量大幅上调,且显著高于热敏自交系CX7-3;根中表达量则在热敏自交系CX7-3中较高,在高温诱导后显著上调。本研究结果为进一步探讨BrHsfA3基因在菜心响应高温胁迫应答中的调控作用奠定基础。
庞强强, 孙晓东, 周曼, 蔡兴来, 张文, 王亚强. 菜心BrHsfA3基因克隆及其对高温胁迫的响应[J]. 生物技术通报, 2023, 39(2): 107-115.
PANG Qiang-qiang, SUN Xiao-dong, ZHOU Man, CAI Xing-lai, ZHANG Wen, WANG Ya-qiang. Cloning of BrHsfA3 in Chinese Flowering Cabbage and Its Responses to Heat Stress[J]. Biotechnology Bulletin, 2023, 39(2): 107-115.
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引物用途Primer purpose |
---|---|---|
BrHsfA3-F1 | CGCCAGAATCATACTCCCGC | 基因合成 Gene synthesis |
BrHsfA3-R1 | CCTTAGAGGACAGAAGCATC | 基因合成 Gene synthesis |
BrHsfA3-F2 | GATGTGCTGCAAGGCGATTA | 基因合成 Gene synthesis |
BrHsfA3-R2 | TTATGCTTCCGGCTCGTATG | 基因合成 Gene synthesis |
BrActin-F | AGCAACTGGGATGACATGGA | RT-qPCR内参基因 RT-qPCR reference genes |
BrActin-R | TCACCAGAGTCGAGCACAAT | RT-qPCR内参基因RT-qPCR reference genes |
Q-BrHsfA3-F | GTCGACATAACAGACGTGGC | RT-qPCR |
Q-BrHsfA3-R | CTGTCTGACGAAGCTGGAGA | RT-qPCR |
表1 基因克隆和表达分析引物
Table 1 Primers used for gene cloning and expression analysis
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引物用途Primer purpose |
---|---|---|
BrHsfA3-F1 | CGCCAGAATCATACTCCCGC | 基因合成 Gene synthesis |
BrHsfA3-R1 | CCTTAGAGGACAGAAGCATC | 基因合成 Gene synthesis |
BrHsfA3-F2 | GATGTGCTGCAAGGCGATTA | 基因合成 Gene synthesis |
BrHsfA3-R2 | TTATGCTTCCGGCTCGTATG | 基因合成 Gene synthesis |
BrActin-F | AGCAACTGGGATGACATGGA | RT-qPCR内参基因 RT-qPCR reference genes |
BrActin-R | TCACCAGAGTCGAGCACAAT | RT-qPCR内参基因RT-qPCR reference genes |
Q-BrHsfA3-F | GTCGACATAACAGACGTGGC | RT-qPCR |
Q-BrHsfA3-R | CTGTCTGACGAAGCTGGAGA | RT-qPCR |
图6 BrHsfA3蛋白二级结构预测 横轴表示氨基酸位置;蓝色区域表示α-螺旋;紫色区域表示无规则卷曲;红色区域表示延伸链;绿色区域表示β-转角
Fig. 6 Prediction of BrHsfA3 protein secondary structure Horizontal axis indicates amino acid position. The blue part indicates alpha helix. The purple indicates random coil. The red part indicates extended strand. The green part indicates beta turn
图9 BrHsfA3基因在高温胁迫处理下的FPKM值 不同小写字母表示同一类型自交系植株差异显著(P< 0.05),下同
Fig. 9 FPKM value of BrHsfA3 gene under heat stress Different lowercase letters of the same inbred indicate significant difference(P<0.05),the same below
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