The Cloning And Function Analysis On MYB21 Gene Related To Anther Development Of Kenaf(Hibiscus Cannabinus L.)

Flower is the reproductive organ of flowering plants, Anther is the important function part of stamen. Anther development is closely related to the ability of plant reproduction and regulated by precision and complexity, especially on the regulation of transcriptional level,transcription factors play an important role in this regulation process. Among the numerous transcription factors, myb transcription factors are one of the largest family of transcription factors in plants, which involved in controlling many kinds of physiological and biochemical processes in plants, some of the myb transcription factors play an important role in regulating the development of pollen and anther, and the abnormal development of anther is usually the visual expression of crop male sterility. Cytoplasmic male sterility is not only the theoretical basis of heterosis utilization in crop, but also the important way to create new crops. Carrying out researches on the relationship between the anther development and transcription factors in kenaf has an important practical significance, because kenaf is one of the most potential multi-purpose crops in the 21st. This experiment screened out the MYB21 gene for specific researches through analyzing the results of the high-throughput transcriptome sequencing data of kenaf, the preliminary results are as follows:1. Extracted high quality total DNA and total RNA of the kenaf anther in male sterility P3A and its maintainer P3B using the improved CTAB method; and taking advantage of homology cloning method, combined with the results of the high-throughput transcriptome sequencing of kenaf, searching the MYB21 gene sequence and finding its longest ORF sequence, and then designing primers from the initiator codon to the termination codon of the longest ORF sequence, taking cDNA and total DNA as template, we successfully cloned the sequence of MYB21 gene and knowed the DNA sequence of the MYB21 gene contains two introns by comparing the cDNA and total DNA level. The Sequence of MYB21 has been submitted to NCBI database, the accession number is KT898146.2. Extracted the total RNA of root, leaf, anther from sterile line and its maintainer line of kenaf, and obtained the identical concentration of cDNA by reverse transcription, taking the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene, designing specific primers of reference gene and target gene according to the principles of qRT-PCR primers, using the Fluorescent dye insertion method to analyse the expression level of MYB21 gene in different organs between the sterile line and its maintainer line of kenaf. The results show that the expression level of MYB21 gene in the anther of sterile line is significantly lower than that of its maintainer line, which is consistent with the results of the high-throughput transcriptome sequencing, so we suggest that MYB21 gene may takes part in the development of anther of kenaf.3. Designed the primers to amplify the sequence which was inserted into the over-expression vector according to the full length of MYB21 gene cDNA sequence, and Adding the corresponding enzyme cleavage site sequence and protective bases to the 5’end of the primers, Make the amplified sequence connected to the transformed PBI121-GFP vector with T4 DNA ligase. We confirmed that the over-expression vector was constructed successfully through enzyme digestion and sequencing;4. According to the principles of RNAi vector construction, we select the specific sequence as interference fragment of MYB21 gene cDNA sequence, and make it connect to the intermediate expression vector pKANNIBAL and plant binary expression vector PART27. The results of enzyme digestion and sequencing confirmed that the RNAi vector was constructed successfully.5. Using pollen-tube pathway transgenic technology, we injected the over-expression vector into the kenaf, and obtained two strains of anther abortion mutant through preliminary phenotypic observation.6. Finally, the over-expression vector and RNAi vector were transferred into Arabidopsis thaliana by agrobacterium mediated inflorescence infection method, and transferred into tobacco by leaf disc transformation. The transgenetic seedlings are being screened.