油菜育性相关多聚半乳糖醛酸酶基因MF6的获得及表达分析

本研究目的在于利用化学杀雄剂“化杀灵WP1”锈导甘蓝型油菜R121产生雄性不育,并对花蕾中的差异表达基因进行分离、筛选及定量表达分析.首先,通过抑制消减杂交技术(SSH)和反向Northern斑点杂交技术,分离和筛选出在雄性不育花蕾中差异表达的基因片段.然后,将差异片段测序后在NCBI数据库中进行同源性比对及功能分析.最后,对差异表达基因进行反转录荧光定量表达(RT-QPCR)分析.本研究从筛选出来的6个阳性克隆中获得一个与多聚半乳糖醛酸酶基因(MF6) 100％同源的基因片段,该基因被认为与花粉生长发育及花粉成熟密切相关.对MF6基因在不同长度及不同育性花蕾中的表达量进行了定量分析,结果显示,MF6基因在雄性可育花蕾中的表达量均高于雄性不育花蕾,且在蕾长1.00 mm～1.50 mm和3.50 mm～4.00mm时分别达到了18.53倍和43.15倍的差异.因此,我们推测“化杀灵WP1”主要通过抑制花粉母细胞减数分裂时期中MF6基因的表达,使花粉产生败育:同时,MF6基因的表达可能也与花丝的伸长生长有关.

The Acquisition and Expression Analysis of Polygalacturonase Gene MF6 Related to the Fertility of Brassica napus L.

The purpose of this study is to analyze the differentially expressed genes in the buds of male sterile plants which were induced by a male sterilizing chemical Hua-Sha-Ling WP1 in Brassica napus L.R121.First,we isolated the differentially expressed gene fragments from the male sterile buds through the suppression subtractive hybridization(SSH) and the reverse northern dot blotting.Then,homology comparisons and functional analysis were conducted through online database NCBI.Finally,the differentially expressed gene was further analyzed through reverse transcription quantitative PCR.In the results,we obtained a gene fragment which is 100% homologous to the gene of polygalacturonase(MF6) in Arabidopsis from the 6 positive clones.It has been reported that the polygalacturonase gene(MF6) is closely related to the development and maturation of pollen.The amount of MF6 gene expression in flower buds with different lengths and different fertilities was analyzed quantitatively.The results show that the expression of MF6 gene in the male fertile flower buds is higher than that in the male sterile flower buds.And the differences are about 18.53 times and 43.15 times when the bud lengths are 1.00 mm～1.50 mm and 3.5 mm～4.0 mm,respectivly.Therefore,Hua-Sha-Ling WP1 presumably inhibited the expression of the MF6 gene during the meiosis stages of pollen mother cell and caused the abortion of pollens.At the same time,the inhibition of MF6 gene expression might also be associated with the elongation of filaments.