Cloning And Expression Pattern Analysis Of A Pollen Development Related Gene BrSKS13 In Chinese Cabbage

In our previous study,a 412bp cDNA fragments was obtained by using the suppression subtractive hybridization(SSH)technology.In order to explore the the length sequences information,expression patterns of the target fragment,and predict its biological function in Chinese cabbage,time and space expression patterns of the gene were analyzed by the homologous gene cloning,qRT-PCR,In situ hybridization,and promoter analysis.The results were showed as following:1.The segment that obtained from Chinese cabbage nuclear male sterile lines of sterility related fragments by SSH technology blast with Chinese cabbage genome-wide comparison,found that the homology with Bra001504 is the highest.The results of sequence alignment analysis showed that Bra001504 obtained through SSH technology from Chinese cabbage is homologous with Arabidopsis thaliana SKS13 gene by Blast in NCBI,so it was named as BrSKS13.2.The sequence of the candidate gene cDNA,1656bp,was obtained by homologous cloning.The largest open reading frame analysis of the whole length sequences of candidate gene found that its expressed products is a secreted protein of 551 amino acids with an estimated molecular weight of 61.87 KDa.A signal peptide sequence at the amino terminus was also identified by SignalP4.0 analysis.The first 22 amino acids at the N-terminus constitute the signal peptide.No obvious transmembrane domain structure was found.The protein also contains three Cupredoxin suprefamily conservative domain structure,but the lack of the specific histidine residues in the first and third conservative domain due to their function lost of combining with copper ion as well as the other members of copper oxidase family.Therefore,the second conservative structure domain hold the ability for combining with copper ions,and thus was named as CuRO-2-AAO-like-1,but without the third structure domain to coordinate with,so it cannot play its biological functions.The sequences of the target protein is higher homologous with ascorbic acid oxidase,but lack of two essential amino acid sites to combine with copper ions,so it is a special kind of ascorbic acid oxidase.3.The evolutionary tree constructed form the homologous sequences showed that the genetic relationship of candidate gene is closet with Arabidopsis SKS13,followed are BrSKS14 in Chinese cabbage,SKS14and a pollen development relative gene in Arabidopsis,the relationships with BrSKS12 in Chinese cabbage and SKS12 in Arabidopsis are farer,then the relationships of the BrSKS13 are more farer away with BrSKS11 in Chinese cabbage and SKS11 in Arabidopsis and the members of SKS family in other specises.Homologous sequence alignment analysis found that the sequences of SKS protein family members are highly conservative,may reach more than 83%,and among these genes,the homology of Arabidopsis SKS13 with candidate gene is the highest.So the candidate gene is thought to be the homologous gene of Arabidopsis SKS13 in the Chinese cabbage,named BrSKS13.4.Expression pattern analysis of BrSKS13 in roots,stems,leafs,buds in fertile and sterile plants of Chinese cabbage using qRT-PCR,found BrSKS13 had a higher expression in buds of fertile plants,and BrSKS13 is associated with the buds development of Chinese cabbage.Then the analysis of the BrSKS13 expresses in different organs such as sepals,petals,stamens,and pistils of fertile buds using qRT-PCR showed that BrSKS13 was higher expressed in stamens than in other organs,which illustrated that BrKS13 is associated with stamen development in Chinese cabbage.Then the fertile buds was separated into ?-? six different levels according the length,and used to analyze the expression level of BrSKS13 using qRT-PCR analysis.The results showed that the expression of BrSKS13 is increasing gradually with the increasing levels of buds,it arrives at the higest when the bud grows to VI stages,and the difference is significant,suggested BrSKS13 participate the development of the anther in Chinese cabbage.5.In situ hybridization was used to analyze the expression of BrSKS13 in ?-? stages of the fertile buds,the results showed that hybridization signals was firstly appeared on the pollen grain of anthers at third stage,and gradually increased according with increasing of the buds level,suggested BrSKS13 may be associated with the development of mature pollen.6.The expression profile analysis of BrSKS13 promoter in Arabidopsis showed that the GUS gene after the promoter of BrSKS13 specific express in Arabidopsis anther,which indicated that BrSKS13 gene is related to the anther development in Chinese cabbage.Synthesize all the results above,BrSKS13 should be a special ascorbate oxidase gene,which contains two conserved domains that lose copper ion binding function and a normal copper ion binding domain.And it has the higher expression in the pollen grains of the buds that opening soon,indicated that it is a pollen-specific expression gene,involved in pollen maturation development.