Isolation And Functional Confirmation Of Pollen Coat Protein Gene BcMF5 And Polygalacturonase Gene BcMF6 In Brassica Campestris

Brassica crops is one of most important crops in agricultural production. The male sterile line was widely utilized in practice to produce F1 hybrid seeds. The research about male sterility mechanism of Brassica contributed to understanding of microspore and pollen development process and provided theory direction to create man-made male sterile line. By cDNA-AFLP technology, two differential fragments named BcMF5 and BcMF6, 179 bp and 346 bp respectively in length, were obtained from the wild type of male meiotic cytokinesis (mmc) mutant (male sterile plant) of Brassica campestris L. ssp. chinensis Markino. Sequence analysis indicated that BcMF5 shared a high homology to pollen coat protein (PCP) genes, while BcMF6 had a high similarity to polygalacturonase (PG) genes. Earlier study had proved that these two fragments specially expressed in middle flower buds, large flower buds and open flowers in the wild type (male fertile plant) of Brassica campestris, while not in mmc mutant at all. This demonstrated that they belonged to the late-expressed gene deciding on pollen fertility. However, their precise biological function were unknown. In this research, firstly, their RNAi or anti-sense RNA expression vector were constructed;secondly, B. campestris L. ssp. chinensis Markino var. parachinensis (Bailey) Tsen et Lee] was transformed by agrobacterium-mediated method to obtain their loss-of-function mutant. Lastly, their function in pollen development process were analyzed. The major study results as follows:(1) On the basis of cDNA-AFLP differential fragments, the cDNA sequence of BcMF5 and BcMF6 were isolated by rapid amplification of cDNA ends (RACE) . Subsequently, their DNA sequences were obtained. Sequence comparison indicated that the largest open reading frame (ORF) of BcMF5 was composed of 252 bp with a deduced 83 amino acids and is interrupted by a 268-bp intron. Homology analysis showed that BcMF5 belonged to one member of PCP-A gene family, shared a conservative characteristic of PCP and played a role in pollen-stigma interaction and pollen recognition during plant sexual production. BcMF6 consists of 1,194 bp encoding a protein of 358 amino acids and is interrupted by three introns of 81 bp, 95 bp and 127 bp in length. Homology comparison and phylogenetic analysis showed that BcMF6 had relation to pollen-expressed PG.(2) The RNAi expression vector of BcMF5 containing constitutive promoter CaMV35s and tapetum-expressed promoter A9, BcA9 were constructed, respectively. By agrobacterium-mediated method, their flower Chinese cabbage transgenic plantlets were obtained.(3) The antisense expression vector of BcMF6 containing constitutive promoter CaMV35s and tapetum-expressed promoter A9, BcA9 were constructed, respectively. By agrobacterium-mediatedmethod, their flower Chinese cabbage transgenic plantlets were obtained.(4) Molecular, morphological and cytological characterization of flower Chinese cabbage transgenic KanR plantlets transformed from pBI35S-RMF5> pBIA9-RMF5 and pBIBcA9-RMF5 were carried out. Using cDNA of BcMF5 as probe, the Northern hybridization results showed that the expression of BcMF5 in stage V flower bud (diameter > 2.8 mm) and open flower of pB135S-RMF5 were sharply inhibited. But its repressibility level to the expression of BcMF5 was less than pBIA9-RMF5 and pBIBcA9-RMF5. On the one hand, this illustrated that BcMF5 had a close relation to tapetum. On the other hand, CaMV35S promoter was proved to be active in flower bud and open flower. With the maturation of pollen, the activity levels of CaMV35S promoter increased. As far as pB135S-RMF5 was concerned, 32.41% pollen germination ratio in vitro and 22.19% pollen abnormality by scan electron microscope illustrated that CaMV35S promoter expressed at a low level in pollen coat, but its expression level enhanced during pollen germination and pollen tube extension. Accordingly, the repression level to BcMF5 increased and pollen germination ratio decreased. Pollen abnormality of pBIA9-RMF5 and pB!BcA9-RMF5 in scan electron microscope were 88.37% and 94.28% respectively, which proved that BcMF5 belonged to PCP, its origin come from tapetum and it was essential for BcMF5 to keep normal shape of pollen. When BcMF5 was inhibited, the development of pollen coat would be captured. Surprisingly, the pollen germination ratio of pBIA9-RMF5 and pBIBcA9-RMF5 were distinct from each other. However, pollen germination assay in stained pistil by aniline blue after pollination 24 h by fluorescence microscope indicated that the expression of BcMFS was inhibited by pBIBcA9-RMF5 just as pBI35S-RMF5 and pBIA9-RMF5, which resulted in adhesion failure of pollen to pistil and germination ratio of pollen decreased.(5) Molecular, morphological and cytological characterization of flower Chinese cabbage transgenic KanR plantlets transformed from pBI35S-AMF6> pBIA9-AMF6 and pBlBcA9-AMF6 were carried out. Using cDNA of BcMF6 as probe, the Northern hybridization was performed in open flower of pBI35S-AMF6^ pBIA9- AMF6 and pBIBcA9-AMF6. As a result of antisense RNA repression, the expression of BcMF6 was caught and decreased sharply in comparison to positive CK, which proved that BcMF6 was a pollen-expressed PG. In addition, the flower organ (decreased pollen number and thin anther) of flower Chinese cabbage transgenic kanR plantlets of pBI35S-AMF6> pBIA9-AMF6 and pBIBcA9-AMF6 were distinct from wild type. This illuminated that BcMF6 act in pollen and CaMV35S promoter had activity in pollen, which was consistent with the study results of chapter 2. In pBI35S-AMF6, 48.67% pollen germination ratio in vitro and 54.67% pollen abnormality by scan electron microscope also confirmed that CaMV35S promoter may act in the early stage of pollen development. Furthermore, the shape of abnormality pollen (shrinked, not fully-developed pollen) was different from pBIA9-AMF6 and pBIBcA9-AMF6, which demonstrated that .BcMF6 played a role during pollen maturation.Although tapetum had dissolved before anthesis and pollen maturation, except for the higher pollen abnormality of pBIBcA9-AMF6, pollen abnormality of pBIA9-AMF6 by scan electron microscope,pollen germination ratio in vitro of both pBIBcA9-AMF6 and pBIA9-AMF6, were all 50% or so, which were distinct from positive control. This tell us that BcMF6 played a role in tapetum, pollen germination and pollen tube extension, but also, it may was gametal expression pattern.(6) Promoter of BcMF5 was isolated by TAIL-PCR for the first time. A promoter region was predicted in its sense strand. In addition, another promoter region was also found in its reverse complementary strand, which showed that promoter of BcMFS may be a bidirectional promoter. Sequence analysis indicated that several pollen-expressed function elements were present in promoter of BcMF5, such as 56/59 box and its variant, AGAAA element which was indispensable to pollen activity of tomato LAT52 promoter and TGTTGGTT, the variant of PB core elements of LAT52/56 box and AAATGA element of modulating pollen activity of ntp303 promoter etc. Furthermore, some elements to respond to environment were also found in sense and reverse complementary strand of BcMF5 promoter. Interestingly, homology search to GENEBANK by blast found a 94-bp fragment in the sense strand of BcMF5 promoter to share 91% homology to its 3'-UTR, which illustrated a interaction mechanism existence between 3'-UTR and 5'-UTR of BcMF5 co-regulated the transcription of PCP.(7) Bidirectional deletion expression vector containing 612-bp and 380-bp promoter sequence of BcMF5 were constructed respectively. By Agrobacterium-mediated method, pBI-S612 and pBI-AS380 were sucessfully transformed into flower Chinese cabbage. The primary function analysis towards flower Chinese cabbage transgenic plantlets of pBI-S612 and pBI-AS380 proved that promoter of BcMF5 was probably a pollen-expressed bidirectional promoter, because both sense strand and reverse complementary strand had an ability to drive GUS expression in pollen. In addition, TCA elements (responsive to salicylinc acid), found in reverse complementary strand of BcMFS promoter, was thinked to enhance tolerance of the transgenic plantlets of pBI-AS380 to environment stress with its high vigor and high fruit-setting ability.(8) Eight different length transcripts of 3'-UTR of BcMF5 were first isolated from the wild type of mmc mutant in B. campestris ssp. chinensis. Based on sequence analysis, using Southern hybridization combined with RT-PCR and Northern hybridization, their temporal and spacial expression profiles were studied. The results indicated that eight different length transcripts should be a result of multiple sites polyadenylation of 3'-UTR of BcMF5;different length transcripts had difference in their temporal and spacial expression patterns;in 3'-UTR, the presence of the A-U rich elements (AREs) maybe was a mechanism that made for renewal and translation efficiency of the mRNA of PCPs. The presence of internal ribosome entry site (IRES) that was usually found in 5'-UTR maybe involved in an interaction mechanism between 3'-UTR and 5'-UTR that co-regulate the expression of PCPs..