Identification And Functional Analysis Of The Gene Family Of ATP-Binding Cassette Transporter In Camellia Oleifera

Camellia oleifera is a kind of woody oil tree with great edible value and economic value in China.Camellia oleifera is a kind of self-incompatibility tree species,which needs to be planted in a reasonable variety allocation.In order to analyze the molecular mechanism of self-incompatibility of Camellia oleifera,the team of our laboratory has constructed a pistil transcriptome database with significant differences between self-pollination and cross-pollination based on the main cultivars of Camellia oleifera,’Huaxin’ and ’Huaj in’.Through transcriptome analysis,we find that the metabolic pathway of ABC transporter is closely related to self-incompatibility of Camellia oleifera.Therefore,we begin to carry out gene cloning and function research of ABC transporter in Camellia oleifera.The results are as follows:1.Cloning and bioinformatics analysis of six ABC transporter genes in Camellia oleifera.Six distinct ABC transporter genes are screened from the transcriptome data of self and cross pistil of Camellia oleifera.Six pairs of specific primers are designed to amplify and clone the full-length CDs gene sequence,which are 3381 bp,2142 bp,2172 bp,1935 bp,2166 bp and 3318 bp,encoding 1126,713,2172,644,721,1105 amino acids respectively.The protein molecular mass are 126.53,79.63,79.93,72.65,80.47,121.84 kDa,with theoretical isoelectric point of 9.02,5.8,8.84,7.91,80.47,8.9.Prediction results show that they have 13,0,6,6,6,5 transmembrane structures.The conserved domain analysis shows that all of the six genes contain NBD domain,including Walker A,ABC tag sequence and Walker B,which are Identified as ABC transporter family.The six genes are named as:CoABCC9,CoABCF3,CoABCG3,CoABCG5,CoABCG11 and CoABCG28.2.The expression patterns of six ABC transporters in different tissues and different periods of Camellia oleifera are revealed.The results showed that there are significant differences in the expression of genes in different tissues.The expression of CoABCC9 and CoABCF3 gene are higher in the old stem and pollen,CoABCG3 and CoABCG5 gene are higher in the old stem and bud,CoABCG11 gene is higher in the petal,and CoABCG28 gene is specifically expressed in the pollen.There are also significant differences in the expression of each gene in the style and ovary at different pollination stages.The expression of CoABCC9,CoABCF3,CoABCG3,CoABCG28 gene in self-pollination style are higher than that in cross and non-pollination at 24 or 36 hours;the expression of CoABCC9,CoABCG3,CoABCG11,CoABCG28 gene at 36 or 48 hours of pollination,the expression of self-pollination ovary is higher than that of cross-pollination and non-pollination.The results show that these genes are significantly different in different tissues and different periods,May be involved in self-incompatibility reaction.3.Based on the cloning of CoABCF3 gene sequence,the prokaryotic expression vector Pcold-TF-CoABCF3 is constructed.The target protein with a molecular weight of 79.93 kDa is successfully induced by transformation into Ecoli(DE3)expression vector.4.Subcellular expression vectors of six genes are successfully constructed.The results show that the genes of CoABCC9,CoABCG11 and CoABCG28 are distributed in cell membrane and chloroplast,CoABCG3 and CoABCG5 are mainly distributed in cell membrane,and CoABCF3 gene is mainly distributed in mitochondria.5.The overexpression vectors of CoABCC9,CoABCF3,CoABCG3,CoABCG5,CoABCG11 and CoABCG28 genes are constructed and transformed into Arabidopsis through by pollen-tube pathway and successfully obtained the T2 generation positive plants.The results of phenotype observation show that the root growth of CoABCC9 gene overexpression plants is significantly longer than that of wild-type plants,and the growth of transgenic leaves is more vigorous than that of wild-type Arabidopsis,the fruit of CoABCG3 gene overexpression plants is significantly shorter than that of wild-type Arabidopsis,the phenotype of other four gene overexpression plants have no significant difference.6.In oder to measure the content of soluble sugar,the flower buds of T2 generation of CoABCC9,CoABCF3,CoABCG28 gene overexpression plants are collected as experimental materials.The results show that the content of soluble sugar in CoABCC9 gene overexpression plants is significantly higher than that in wild type Arabidopsis,the content of soluble sugar in CoABCF3 gene overexpression plants is significantly lower than that in wild type Arabidopsis,the content of soluble sugar in CoABCG28 gene overexpression plants is lower than that in wild type Arabidopsis.