棉花抗细胞凋亡基因GhDAD1的克隆、定位及表达分析

Chromosome Mapping and Expression Analysis After the Cloning of Defender Against Apoptotic Cell Death Gene from Gossypium hirsutum

【目的】克隆陆地棉抗细胞凋亡新基因GhDAD1,为陆地棉细胞凋亡的分子机制提供依据,为培育不早衰陆地棉品种提供理论基础。【方法】采用RT-PCR以及电子克隆获得陆地棉GhDAD1的基因组序列以及全长cDNA序列并进行生物信息学分析,然后通过荧光原位杂交(FISH)技术进行染色体定位,利用real-timePCR进行表达模式分析,分析6-BA、乙烯、H2O2、SA以及NO对GhDAD1表达量的影响。【结果】棉花GhDAD1编码阅读框全长354bp,包含5个外显子,4个内含子以及232bp的5′非编码区和280bp的3′非编码区。氨基酸序列分析表明,GhDAD1蛋白属于DAD家族,与柑桔、拟南芥GhDAD1蛋白的相似性分别为91%和88%,起始密码子区符合Kozark规则,内含子剪接位点符合GT-AG规则。FISH技术将GhDAD1定位于染色体长臂上。real-time PCR分析表明,陆地棉各组织中均表达该基因,花和种胚等幼嫩组织表达量较高,并且随着衰老的进行,表达量降低。利用6-BA、乙烯、水杨酸、一氧化碳以及双氧水处理中棉所10号,qRT-PCR分析表明,6-BA、水杨酸处理能够延缓衰老,增加GhDAD1的表达量;乙烯能够加速衰老,降低GhDAD1的表达量;H2O2对GhDAD1的表达量的影响不大;而NO不同浓度影响不一样,随着浓度的升高,GhDAD1的表达量先升高后降低【。结论】陆地棉中存在抗细胞凋亡基因(GhDAD1)。

Objective Cloning a defender against apoptotic cell death gene,GhDAD1,and analyzing its expression model could provide a theoretical foundation for both molecular mechanism research of apoptosis and breeding senescence-resistant varieties in shorted-season Gossypium hirsutum.Method RT-PCR and in silico cloning were used to amplify DNA sequence and full-length cDNA sequence of GhDAD1 in Gossypium hirsutum.Bioinformatics characterization of GhDAD1 had also been analyzed through all kinds of software.FISH was used to get the location of GhDAD1 on chromosomes.Both the expression patterns of GhDAD1 under the nature decrepitude and treatment of 6-BA,ethylene,H2O2,SA,NO were analyzed by real-time PCR.Result The length of GhDAD1 sequence is 866 bp,including a 354 bp ORF,232 bp 5＇-UTR,280 bp 3＇-UTR,five extrons and 4 introns.It was in conformity with the principles of Kozark and GT-AG.Homology analysis showed that the GhDAD1 was highly homologous to other DAD1 from different species,especially from Populus davidiana,Citrus unshiu（91%） and Arabidopsis thaliana（88%） other than barley and rice.GhDAD1 protein had 112 amino acids,and its pI was 8.32.The GhDAD1 gene was located on the long arm of the chromosomes.Real-time PCR showed that GhDAD1 gene expressed in all issues of Gossypium hirsutum,with flower and embryo having high expressing level.And the expressing level was on the decline with the processing of decrepitude.The CCRI10 was cultured by addition of 6-BA,ethylene,SA,NO,and H2O2.At the same time,chlorophyll was measured.The result of qRT-PCR showed that 6-BA and SA could retard the senescence,and the transcript of GhDAD1 was higher.The ethylene could accelerate the senescence,so the transcript of GhDAD1 was lower.The influence of H2O2 was not apparent.With the increasing of NO concentration,the expression level of GhDAD1 increased to a peak then decreased.Conclusion There seemed to be a gene about the defender against apoptotic cell death 1 in Gossypium hirsutum.