辣椒乙烯反应抑制因子Cacl-6468的克隆及其抗根结线虫作用分析

Cloning of Pepper Ethylene-Responsive Proteinase Inhibitor Cacl-6468 and Its Effect on Resistance to Meloidogyne incognita

Cacl-6468是新发现的辣椒乙烯反应抑制因子基因,来源于辣椒(Capsicum annuum)抗根结线虫材料HDA149,属于抑制因子Ⅰ超基因家族基因(InhibitorⅠ),与马铃薯的乙烯反应抑制因子具有较近的遗传同源关系。在抗性辣椒中Cacl-6468的表达与南方根结线虫早期侵染紧密相关,在毒性线虫侵染12 h时表达量即明显下调,到7 d时达到最低,而在非毒性线虫侵染12 h时则显著上调,3 d时达到最高。利用病毒诱导的基因沉默(VIGS)技术,证实抑制Cacl-6468的表达能促使南方根结线虫非毒群体克服辣椒HDA149的抗性,形成根结和卵块,但其发病程度显著低于毒性线虫。在VIGS测试中还证实Cacl-6468负调控乙烯信号通路,其表达下降伴随着乙烯信号通路基因的显著上调,但是对水杨酸信号通路影响不显著。通过早期病理观察及二龄线虫趋性测试,证实抑制Cacl-6468的表达可以增强辣椒根系对南方根结线虫的吸引性,促进早期侵染。利用气相质谱法检测乙烯释放量发现,抑制Cacl-6468的表达可以增强辣椒植株的乙烯释放量。通过以上结果可推测辣椒乙烯反应抑制因子基因Cacl-6468通过调控乙烯信号通路参与辣椒对根结线虫的抗性调控,抑制该基因表达可以增加辣椒对线虫的感病性,降低抗性。

The pepper gene Cacl-6468 is a novel ethylene-responsive proteinase inhibitor gene,from the nematode-resistance pepper HDA149(Capsicum annuum),belongs to the Potato InhibitorⅠsupper gene family,and has close genetic homology with potato ethylene-responsive proteinase inhibitor.The expression levels of Cacl-6468 in HDA149 were significantly affected by the population kinds of Meloidogyne incognita.For virulence population of M.incognita,Cacl-6468 was down-regulated significantly after 12 h infection,and reached the lowest level at seven days;while for avirulence population,the expression level was significantly up-regulated after 12 h infection,and reached the highest level at three days.Using virus-induced gene silencing(VIGS)technique,it was proved that the knock-down of Cacl-6468 could promote avirulent population to overcome the resistance of pepper HDA149,and developed some galls and egg masses,but the numbers were significantly lower than that of virulent population.The VIGS test also confirmed that Cacl-6468 negatively regulated ethylene signaling pathway,but had no significant effect on salicylic acid signaling pathway.In addition,the early pathological observation and chemotaxis test showed that VIGS treatment of Cacl-6468 enhanced the attraction of pepper root to M.incognita,and promoted early stage infection.The ethylene release of pepper was detected by GC-MS,showed that inhibition of Cacl-6468 expression could enhance the ethylene release of pepper.qPCR results suggested that Cacl-6468 is involved in the resistance regulation of pepper to M.incognita through ethylene signaling pathway,decreased the expression level of Cacl-6468 can increase the susceptibility,and weaken the resistance of pepper HDA149.