Isolation And Functional Confirmation Of BcMF13 And BcMF14 Related To Pollen Development In Chinese Cabbage-pak-choi

Cruciferae crops is one of the vegetable crops species of the highest yield and cultivated most widely as well as is a kind of crops of the most successful in utilizing of heterosis in China. In recent years, an increasingly amount of research has been devoted to the molecular biology of research on the plant breeding of the male sterile line and the basis for application in Brassica crops. However, the mechanism of the plant male sterility is not demonstrated adequately. A lot of questions remain unknown. The cloning and characterization of pollen development related genes is important to analysis the mechanism of male sterility in Cruciferae and construct male sterility line. Along with the development of molecular biological theories and methods, it can provide powerful tools to study on the molecular mechanism of plant male sterility and drive the heterosis to apply in the production in Brassica crops and others. Recently, by cDNA-AFLP technology, two differential fragments named A8T4 and A9T15, 191 bp and 201 bp respectively in length, were obtained in our lab from the wild type of male meiotic cytokinesis (mmc) mutant (male sterile plant) of Brassica campestris L. ssp. chinensis Markino (syn. B. rapa ssp. chinensis). The genes which the two fragments belonging to were named BcMF13 and BcMF14. Earlier study had proved that BcMF13 was a new gene while BcMF14 shared a high homology to Rapid Alkalinization Factor (RALF) genes. In this study, the full length cDNA and DNA of pollen development related BcMF13 and BcMF14 gene was cloned and characterized from Chinese cabbage pak-choi (B. campestris L. ssp. chinensis Makino). Homologous genes with the BcMF13 and BcMF14 were cloned from deferent species in deferent genera of Cruciferae by PCR amplification. Phylogenetic trees of BcMF13 and BcMF14 gene were constructed based on the alignment of their homologous genes, respectively. They provided important molecular data for the study of Cruciferae evolution. Meanwhile, RNAi or anti-sense RNA technology were applied to transforme B. campestris L. ssp. chinensis Markino var. parachinensis (Bailey) Tsen et Lee by agrobacterium-mediated method to obtain their loss-of-function mutant to analyze BcMF13 and BcMF14 function in pollen development process. The major study results were as follows:(1) On the basis of cDNA-AFLP differential fragments A8T4 and A9T15, the cDNA sequence of BcMF13 and BcMF14 were isolated by rapid amplification of cDNA ends (RACE) . Subsequently, their DNA sequences were obtained. Sequence comparison indicated that the BcMF13 cDNA was 600 bp and was interrupted by a 106-bp intron. The largest open reading frame (ORF) of BcMF13 was composed of 222 bp with a deduced 73 amino acids. Homology analysis showed that BcMF13 shared no similarity with any known genes. So BcMF13 was a new gene. BcMF13 exclusively expressed in stage four and five flower buds, open flowers and silique of fertile line, strongly expressed in stamens with no expression in sterility. And many cDNA sequences from the ESTs share high similarity with BcMF13 all came from the generative organs in Brassica. So we suggested that BcMF13 most possibly had a role in pollen development of Brassica. ORF of BcMF14 was composed of 240 bp with a deduced 79 amino acids. Homology comparison and phylogenetic analysis showed that BcMF14 had relation to rapid alkalinization factor gene (RALF). The BcMF14 amino acids had the characteristic of RALF proteins.(2) BcMF13 Homologous genes were isolated from 8 species of Cruciferae by PCR amplification. The identification ratio of nucli acid and amino acid were 80.0% ~ 96.9% and 96.7% ~ 99.9%.The Clustal X program was used to align the homologous sequences from different 8 species of Cruciferae and to produce NJ, ME and MP phylogentic trees based on distances estimated by the two-parameter method with ] 000 bootstrap samples using MEGA 2;1. The result shows three kinds of phylogentic trees have the strictly accordant topologic structure which means the BcMF13 molecular tree can reflect the evolution of BcMF13.(3) BcMF14 Homologous genes were isolated from 17 species of Cruciferae by PCR amplification. The increased the RALF gene families into some new species. The entification ratio of nucli acid and amino acid were high. The proteins they coded all had the characteristic of RALF: one conserved 'YIXY' region and four conserved C residues. These indicated the BcMF14 evolution was slow and it maybe has important role in Cruciferae.(4) The antisense expression vector and RNAi expression vector of BcMF13 containing constitutive promoter CaMV35S were constructed, respectively, named pBI35S-AMF13 and pBI35S-RMF13. By agrobacterium-mediated method, their flowering Chinese cabbage transgenic plantlets were obtained. The antisense expression vector of BcMF14 containing constitutive promoter CaMV35S and tapetum-expressed promoter BcA9 were constructed, respectively, named pBI35S-AMF14 and pBIBcA9-AMF14. By agrobacterium-mediated method, their flowering Chinese cabbage transgenic plantlets were obtained.(5) Using cDNA of BcMF13 as probe, the Northern hybridization results showed that the expression of BcMF13 in pBI35S-AMF13 and pBI35S-RMF13 transforments was sharply inhibited. And the expression of BcMF13 in pBI35S-RMF13 transforments was much lower than the pBI35S-AMF13 transforments. It suggested that the RNA interference technology was better than antisense technology on the BcMF13 gene in Brassica. Investigation of pollen morphology of transgenic plants of flower Chinese cabbage obtained from BcMF14 anti-sense RNA vector by transition electron microscope indicated that when BcMF13 was depressed pollen development got abnormal early in meiosis. The microspore and tapetum were affected. All these greatly reduced the number of mature pollens. As far as pBI35S-AMF13 was concerned, 34.23% pollen germination ratio in vitro and 54.66% pollen abnormality by scan electron microscope. This is more serious in the RNA interference technology, with 26.27% pollen germination ratio in vitro and 57.55% pollen abnormality in the pBI35S-RMF13. Whether pollen germination was normal or not should be confirmed by further in pistil experiment. Moreover, pollen germination in stained pistil by aniline blue after pollination 24 h by fluorescence microscope was examined. The results also indicate that the expression of BcMF13 was inhibited by the two methods, which resulted in adhesion failure of pollen to pistil and germination ration of pollen accordingly decreased.(6) Using 300-bp cDNA of BcMF14 as probe, the Northern hybridization was performed in flower buds of pBI35S-AMF14 and pBIBcA9-AMF14. The result showed that the expression of BcMF14 was caught and decreased sharply in comparison to positive CK, and the expression in pBIBcA9-AMF14 was more decreased than in pBI35S-AMF14, which proved that BcMF14 was a pollen-expressed RALF. This illuminated CaMV35S promoter had activity in pollen, and the activity was less than BcA9, which was consistent with the study results of our lab's transition electron microscope experiments and tissue investigation showed that BcMF14. worked during tetrad. It worked on microspore and tapetum and might have a role in the interaction between the two kinds of cells. In pBI35S-AMF14,48.95% pollen germination ratio in vitro and 26.09% pollen abnormality by scan electron microscope also confirmed that CaMV35S promoter acted in pollen, which was 51.2% and 44.39% in pBIBcA9-AMF14. Moreover, pollen germination in pistil was examined. The results also indicate that less pollen tube could get into the bottom of the pistil on contrast to the control. This tells us that the antisense technology could decrease the expression of BcMF14 and made pollen development abnormal, with many freaky pollens and the co-identity between the pollen and the pistil was affected. All these indicated that in the RALF families there were some signal molecule had certain role in pollen development. These inosculated with Bedinger's discovering, the over-expression or depress of BcMF14 and PRALF could disturb pollen development and restrain the pollen tubes' growing remarkably.