棉花蕾铃离层脱落相关基因的克隆及其表达分析

Cloning and expression analysis of genes related to abscission of cotton boll abscission layer

  • 摘要: 目的研究激素与棉花蕾铃脱落的关系,为解析离层脱落相关基因的表达、调控及功能鉴定提供基础资料.方法以50 mg/L赤霉素(GA3)和250 mg/L乙烯利(CEPA)分别处理的棉花蕾铃离层cDNA为Tester,H2O处理的棉花蕾铃离层cDNA为Driver,利用抑制消减杂交(SSH)技术构建经GA3和乙烯处理后的消减文库,反向Northern blo-tting筛选出离层差异表达的EST序列,然后进行基因功能注释与功能分类,并分析蕾铃离层差异表达基因的组织表达模式和GA3诱导表达规律.结果以GA3处理的离层cDNA为Tester、H2O处理的离层cDNA为Driver,所构建的cDNA文库命名为GA文库;以CEPA处理的离层cDNA为Tester、H2O处理的离层cDNA为Driver,所构建的cDNA文库命名为CE文库.从构建的两个消减文库(GA和CE)中共筛选出29个在离层差异表达的EST序列,通过基因功能注释与功能分类,可将这些EST序列分为翻译相关、代谢相关、信号传导、转录相关、蛋白合成、能量代谢、抗逆相关、基因组序列和未知功能等九大类,其中与翻译相关的EST序列占24.14%,说明经激素处理后棉花蕾铃离层有大量的蛋白合成.从消减文库中挑选10个差异表达基因(EST序列)进行RT-PCR分析,结果发现以GA3处理棉花蕾铃离层后部分相关基因的表达模式会发生变化.结论棉花蕾铃离层经激素处理后能激活脱落相关基因的表达,进而影响器官脱落,是其对逆境胁迫反应的结果.

     

    Abstract: ObjectiveThe present study investigated the relationship between hormones and abscission of cotton boll to provide basic data on gene expression,regulation and function of abscission related genes.MethodThe cDNA of boll abscission layers treated by 50 mg/L gibberellin solution(GA3)and 250 mg/L ethephon solution(CEPA)respectively was used as Tester,while cDNA prepared from abscission layers which were treated with H2O was used as Driver. Subtractive library treated with GA3 and CEPA was constructed by suppression subtractive hybridization(SSH)technology and the EST sequence differentially expressed in abscission layer was selected by reverse Northern blotting. Then gene function annotation and function classification were carried out. The tissue expression patterns and GA3 induced expression patterns of the differentially expressed genes in boll abscission layer were analyzed.ResultThe Tester was the cDNA prepared from abscission layers treated with GA3,the Driver was the cDNA prepared from abscission layers treated with H2O. The constructed library was named GA library. The Tester was the cDNA prepared from abscission layers treated with CEPA, the Driver was the cDNA prepared from abscission layers treated with H2O. The constructed library was named GA library. Twenty-nine EST sequences differentially expressed in abscission layer were found in the two constructed subtractive li-braries(GA and CE)by sequence analyzed. Through gene function annotation and function classification,the EST se-quences were involved in translation,metabolism,signal transduction,transcription,protein synthesis,energy metabo-lism,stress defense,genome DNA and unknown function. The genes that related to translation accounted for 24.14%, which showed that there was a great deal of protein synthesis after hormonal treatment in abscission layer cells. Ten diffe-rentially expressed genes(EST sequence)from these libraries were analyzed by RT-PCR. It was found that the expression patterns of some genes changed after the cotton boll abscission layer was treated with GA3.ConclusionAfter hormone treatment,the abscission layer of cotton boll can activate the expression of abscission-related genes,thus affecting the ab-scission of organs,which is the result of their response to adversity stress.

     

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