海岛棉GbRLCK10基因克隆及表达分析

Cloning and Expression Analysis of the GbRLCK10 Gene in Gossypium barbadense L.

该研究以拟南芥抗逆基因At1g67520为探针,利用海岛棉ESTs数据库,通过电子克隆获得海岛棉RLCK家族基因GbRLCK10,解析该基因组结构,并结合qRT-PCR技术分析该基因mRNA的组织表达特征以及在不同胁迫诱导下的表达模式,为揭示RLCK家族基因在海岛棉中的表达调控及作用机制提供理论依据。结果显示:(1)获得海岛棉类受体胞质激酶(RLCK)基因,其开放阅读框(ORF)为1 179bp,编码392个氨基酸,具有典型的Serine/Threonine结构域,属于RLCK家族,与GaRLCK10(XP_017604046.1)亲缘关系较近,命名为GbRLCK10(登录号2022184),且该基因由5个外显子和4个内含子组成。(2)实时荧光定量(qRT-PCR)检测显示,GbRLCK10基因在抗病品种‘新海21’和感病品种‘新海14’的根、茎、叶中均有表达;当黄萎病菌诱导后,GbRLCK10基因在抗病品种中对于病原菌的响应时间早于感病品种,且对黄萎病菌响应更强烈,推测该基因参与棉花对黄萎病的响应;盐(NaCl)、干旱(PEG-6000)处理‘新海21’后,GbRLCK10基因在NaCl处理下响应时间要早于PEG-6000处理,但对PEG-6000处理响应更强烈;分别用4种激素处理‘新海21’后,GbRLCK10均能被诱导表达,且在水杨酸(SA)处理后表现为先增加后下降再增加趋势,在乙烯(ET)处理后表达量为持续上升趋势,在茉莉酸甲酯(MeJA)处理后呈先升高然后下降的趋势,但GbRLCK10基因对赤霉素(GA3)响应不明显。研究表明,GbRLCK10基因具有RLCK基因家族典型特征,该基因随黄萎病菌、NaCl、干旱、激素处理时间推移而发生变化,推测GbRLCK10基因可能参与了棉花对黄萎病菌、NaCl、干旱、激素胁迫的应答反应,但其功能仍需进一步研究。

In this study,the anti-stress A t1g67520gene from Arabidopsis thaliana was used as a probe,a RLCK gene of GbRLCK10was cloned through the in silico cloning and RT-PCR.To reveal the expression,regulation and mechanism of RLCK family genes in Gossypium barbadense,we has analyzed the genomic structure and the expression pattern of GbRLCK10by combining with qRT-PCR technology under mRNA organization and different stress inducements.Results show that:(1)the ORF of GbRLCK10was1179bp,encoding392amino acids.GbRLCK10contained only the Serine/Threonine kinase domain,and belonged to receptor-like cytoplasmic kinase gene family.Phylogenetic analysis showed that GbRLCK10closed to GaRLCK10and named as GbRLCK10(Genebank:2022184).The gene was made up of5exons and4introns.(2)The GbRLCK10expression were detected by qRT-PCR,the results showed that GbRLCK10gene is expressed in root,stem and leaf of resistant cultivar‘Xinhai21’and susceptible cultivar‘Xinhai14’.With the duration of the treatment of Verticillium dahliae,while the relative expression of GbRLCK10was significantly lower and the response was later in the susceptible cultivar than that in resistant cultivar.Both NaCl and PEG6000could induce the expression of GbRLCK10,the expression of GbRLCK10increased firstly and decreased.After resistant cultivar‘Xinhai21’was treated by NaCl,response time of GbRLCK10is earlier than PEG6000,but the response to PEG6000was more intense.Salicylic acid,ethylene,methyl jasmonate and gibberellic acid could also induce the expression of GbRLCK10.After the treatment of salicylic acid,the level of GbRLCK10expression increased and decreased,then increased again.The expression of GbRLCK10increased and maintained at a high level when induced by ethylene.However,the expression of GbRLCK10firstly increased and then decreased when induced by methyl jasmonate.Compared with salicylic acid,ethylene,methyl jasmonate,the gibberellic acid induced expression levels of GbRLCK10were significantly lower.We suggested that GbRLCK10may be response to pathogens,NaCl,PEG6000and hormones stress in Gossypium barbadense L.,but it s function still need further study.