Cloning, Expression And Functional Confirmation Of BcMF11 And BcMF12 Related To Pollen Development In Brassica Campestris Ssp. Chinensis

Brassica crops are one of most universal crops in utilizing of heterosis successfully with wide distribution and rich variety resources. It is very important for producing F₁ hybrid seeds to collecting and creating the male sterility line. The isolation and functional analysis of the genes related to pollen development was carried out, so as to further understand the molecular mechanism of pollen development in Brassica and construct man-made male sterile line by reverse genetics technology. In this study, BcMF11 (Brassica campestris male fertility gene 11) and BcMF12(Brassica campestris male fertility gene 12) gene related to pollen development was cloned and characterized from Chinese cabbage (Brassica campestris L. ssp. chinensis Markino, syn. B. rapa ssp. chinensis). The expression pattern of the two genes was analyzed by Northern and RT-PCR(reverse transcriptase polymerase chain reaction) during the different stages of plant development. Then the plant antisense expression vectors of BcMF11 and BcMF12 were constructed regulated by the constitutive promoter CaMV35S(Cauliflower mosaic virus 35S) and tapetum-expressed promoter BcA9(Brassica campestris A9). The transgenic plants were obtained successfully by genetic transformation. Finally, molecular, morphological and cytological characteristics of the pollen were studied in transgenic Kanamycin resistant plants, in order to elucidate the relation of the two genes and pollen development, and understand the systematic regulation and molecular mechanism of pollen development in Brassica. The results were as follows:(1) The full-length of BcMF11(DQ925484) has been cloned from Chinese cabbage using rapid amplification of the cDNA ends (RACE). The BcMF11 cDNA has a total length of 828 bp with multiple termination codons and poly (A) tail, but no prominent open reading frame (ORF). The sequence of BcMF11 had a high A/T content of 58.7% and the minimum free energy of the structure was considerably lower, only -203.26 kcal·mol^-1. No significant similarities were observed between BcMF11 and previously published sequences in GenBank. Analysis of the sequence demonstrated that BcMF11 is a novel non-coding RNA. At the same time, we found some as-acting elements in the sequence of BcMF11, which may be responsible for enhancing the pollen-specific expression of BcMF11.Northern blot and RT-PCR analysis indicated that BcMF11 was expressed in the flower bud, flower and anther. Furthermore, BcMF11 mRNA transcript increased gradually with the pollen development. So BcMF11 is a novel pollen-specific ncRNA and may be involved in the pollen development of Chinese cabbage. (2) BcMF11 homologous genes were isolated from 10 species in Brassica by PCR amplification. Analysis of the sequence found that BcMF11 Homologous genes contain the common characteristics of non-coding RNA, which indicates the nucleotides of non-coding RNA are conservative in the evolution of Brassica. The NJ phylogenetic tree was constructed on the basis of the homologous sequences aligning from different 10 species in Brassica. And the distance was estimated by the two-parameter method with 1000 bootstrap samples. The results showed that B. campestris pekinensis (Huangya 14) and B. campestris chinensis (Youqing) form a specific clade, and B. compestris pekinensis (Xiaoqingkou) and B. campestris rapifera (Wenzhou-pancai) belong to a different clade.(3) BcMF12(DQ925483) was isolated from Chinese cabbage using RACE based on a pollen-specific cDNA fragment BBP1. The cDNA was 1155 bp in length with an ORF of 894 bp capable of encoding a putative polypeptide of 297 amino acids. Secondary structure composition prediction showed that there were 60.61% helix and 35.69% loop in the protein. Furthermore, six highly conserved transmembrane helices were predicted in the deduced amino acid sequence of BcMF12. A blast search revealed that high similarities were found between BcMF12 and some transmembrane protein sequences previously published in the public database, which indicates that BcMF12 may be a novel membrane protein.Northern blot and RT-PCR analysis indicated that the transcripts of BcMF12 were abundant in the middle and big flower bud, flower and anther, which suggest that BcMF12 is a pollen-preferentially gene expressed at the late stages of pollen development.(4) The plant antisense expression vectors of BcMF11 and BcMF12 were constructed containing pBI35S-BcMF11, pBI35S-BcMF12, pBIBcA9-BcMF11 and pBIBcA9-BcMF12 regulated by the constitutive promoter CaMV35S and tapetum-expressed promoter BcA9, respectively. Then these expression vectors were introduced into flowering Chinese cabbage(B. campestris ssp. chinensis var. parachinensis Tsen et Lee) by agrobacterium-mediated method. At last, 61 kanamycin resistant shoots and 305 regenerated plantlets were obtained successfully.(5) Southern and Northern blot was used to test transgenic BcMF11 and BcMF12 Kan^R plantlets on the level of integrating and transcription after PCR screening. The results reveal that the expression of BcMF11 and BcMF12 gene in pollen was down-regulated significantly as the antisense fragments were integrated to transgenic plants. X-Gluc histochemistry staining also show that the antisense fragments has been transcripted and expressed in transgenic flowering Chinese cabbage.(6) The morphologic observation of flower organs in BcMF11 transgenic flowering Chinese cabbage revealed that there are short flower filaments, brown anthers and little pollen in plants. The comparison of the microspore morphology detected by the scanning electron microscope between the transgenic plants and the control plants showed that most of pollen was aberrant in transgenic plants containing pBI35S-BcMF11 and pBIBcA9-BcMF11, and the rate of aberrant microspore reached 88.59% and 91.26%. The cellular observations for pollen development by histochemistry methods revealed that the shape of pollen was abnormal and shrink, the tapetum degenerated and the pollen development was affected. At the same time, the pollen of transgenic plants couldn't germinate normally. The pollen germination ratio in vitro was 31.35% and 37.24% in the transgenic plants containing pBI35S-BcMF11 and pB\BcA9-BcMF11. Therefore, the inhibition expression of BcMF11 will interfere with the pollen formation and development.(7) The morphologic observation of flower organs in BcMF12 transgenic flowering Chinese cabbage revealed that there are white anthers and little pollen in plants. The microspore morphology detected by the scanning electron microscope showed that the rate of aberrant microspore was 89.57% and 23.42% in transgenic plants containing pBIBcA9-BcMF12 and pBI35S-BcMF12. Correspondingly, the pollen germination ratio in vitro was 38.26% and 78.91% in the transgenic plants containing pBIBcA9-BcMF12 and pBI35S-BcMF12. This showed that the activity of tapetum specific promoter BcA9 is stronger than CaMV35S for the regulation of antisense BcMF12 in anthers. Moreover, pollen germination in vivo also indicated the adhesion failure of pollen to pistil and decreased pollen germination rate. So BcMF12 may play an important role in the pollen germination and pollen tube extension as a pollen specific gene in Chinese cabbage.