中国农业科学 ›› 2021, Vol. 54 ›› Issue (20): 4274-4285.doi: 10.3864/j.issn.0578-1752.2021.20.003
薛仁风1(),丰明1,黄宇宁1,Matthew BLAIR2,Walter MESSIER3(
),葛维德1(
)
收稿日期:
2021-04-06
接受日期:
2021-05-27
出版日期:
2021-10-16
发布日期:
2021-10-25
通讯作者:
Walter MESSIER,葛维德
作者简介:
薛仁风,E-mail: 基金资助:
XUE RenFeng1(),FENG Ming1,HUANG YuNing1,Matthew BLAIR2,Walter MESSIER3(
),GE WeiDe1(
)
Received:
2021-04-06
Accepted:
2021-05-27
Online:
2021-10-16
Published:
2021-10-25
Contact:
MESSIER Walter,WeiDe GE
摘要:
【目的】分析普通菜豆PvEG261的序列及表达模式特征,并研究其抗枯萎病和抗旱功能,为普通菜豆镰孢菌枯萎病抗病和抗旱信号调控网络解析及分子育种奠定基础。【方法】对PvEG261开放读码框(open reading frame,ORF)进行生物信息学分析,预测该基因编码蛋白质的理化性质、二级结构、信号肽序列,在NCBI中通过BLASTP检索高同源性蛋白序列进行序列比对并构建系统发育进化树;利用qRT-PCR技术分析PvEG261组织表达特异性及响应枯萎病原菌、干旱胁迫的表达模式;构建PvEG261过表达载体,转化发根农杆菌K599菌株,诱导普通菜豆产生转基因不定根系,同时构建PvEG261沉默载体,其体外转录产物接种普通菜豆,干扰PvEG261的表达,通过接种镰孢菌枯萎病原菌和干旱处理,观察对照、过表达和基因沉默菜豆植株的表型,进行抗病性和抗旱性鉴定,并测定过氧化氢(H2O2)含量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性等生理生化指标。【结果】PvEG261的cDNA序列长471 bp,编码156个氨基酸组成的蛋白质。结构预测其含有10个strand结构,基因编码产物预测分子质量为38.89 kD,理论pI为5.21。PvEG261属于Dirigent超家族成员,包含10个氨基酸的信号肽序列,属于外分泌蛋白。PvEG261与豇豆DIR22蛋白亲缘关系最近,达到91.61%。qRT-PCR结果显示,接种枯萎病原菌和干旱胁迫后,该基因的在菜豆根组织中表达量明显上升,而且该基因具有明显的组织表达特异性,在根中的表达量最高。接种病原菌和干旱胁迫后,与对照相比,过表达植株的抗病性和抗旱性水平明显提高,植株枯萎病发病程度及缺水造成的萎蔫程度均显著降低,根中H2O2含量、POD活性、SOD活性均显著高于对照植株,而MDA含量显著低于对照植株,而基因沉默植株发病程度及萎蔫程度均显著升高,根中H2O2含量、POD活性、SOD活性均显著低于对照植株,MDA含量则显著高于对照植株。【结论】PvEG261响应枯萎病原菌侵染和干旱胁迫,并且正向调控普通菜豆镰孢菌枯萎病抗性和抗旱性水平。
薛仁风,丰明,黄宇宁,Matthew BLAIR,Walter MESSIER,葛维德. PvEG261对普通菜豆镰孢菌枯萎病抗性和抗旱性的影响[J]. 中国农业科学, 2021, 54(20): 4274-4285.
XUE RenFeng,FENG Ming,HUANG YuNing,Matthew BLAIR,Walter MESSIER,GE WeiDe. Effects of PvEG261 Gene on the Fusarium Wilt and Drought- Resistance in Common Bean[J]. Scientia Agricultura Sinica, 2021, 54(20): 4274-4285.
表1
本试验所用引物"
引物名称 Primer name | 引物序列 Primer sequence (5′-3′) |
---|---|
EG261-F | GTTGTGGGAAGTGCTGAA |
EG261-R | CCCTGGCAAATCTGAATA |
ACT-F | GAAGTTCTCTTCCAACCATCC |
ACT-R | TTTCCTTGCTCATTCTGTCCG |
OE-F | GCTCTAGAATGTCCTTAAGCTACAAGAA |
OE-R | GCGAGCTCTTAGTGGTAAACGTAGACGT |
GS-F | GCGGATCCCTTAGCCACTTCAGGTTCT |
GS-R | GCATCGATTCGTGTTGTTCCTCGTTT |
图1
PvEG261序列分析 A:PvEG261蛋白序列分析;B:PvEG261蛋白和其他植物中的Dirigent蛋白序列比对。绿色代表相似性达50%以上;粉色代表相似性达75%以上;黑色代表相似性达100%;C:PvEG261蛋白和其他植物中的Dirigent蛋白的同源进化树分析。CaDIR22:咖啡,XP_027102329.1;LaDIR22:羽扇豆,XP_019462472.1;MpDIR3:刺毛黧豆,RDX73385.1;ApDIR22:相思子,XP_027337739.1;GmDIR22:大豆,XP_006576480.1;GsDIR:野生大豆,KHN26491.1;VaDIR22:红豆,XP_017441835.1;VrDIR22:绿豆,XP_014492698.1;VuDIR22:豇豆,XP_027906892.1"
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