Cloning And Expression Of Energy Metabolism Related Genes And Construction Of RNAi Vector In Kenaf(Hibiscus Cannbainus L.) CMS Line And Maintainer Line

Kenaf (Hibiscus cannabinus L.) belong to Malvaceae Hibiscus, which is an annual bast fiber crop with multiple use. Kenaf has a strong heterosis, and the main way of heterosis utilization is to cultivate kenaf varieties by using the cytoplasmic male sterile line of Kenaf. Therefore, revealing the mechanism of kenaf CMS has an important theoretical significance and a practical value.Numerous studies show that the energy short supply during the period of microspore development of anther lead to the occurrence of the CMS. Currently, the molecular mechanisms of metabolic differences CMS Energy gene expression studies in kenaf focus on the mitochondrial-related genes, while, energy metabolism in the plant nucleus and cytoplasm is a coordinated process, So the study of nuclear energy metabolism differentially expressed genes in male sterile line(P3A) and maintainer line (P3B) is a very important to understand that the mechanism of CMS. Through the analysis of DNA microarray and Kenaf(Hibiscus cannabinus L.) anther transcriptome sequencing data, we found the pentose phosphate pathway key enzyme gene6-PGDH(6-phosphogluconate dehydrogenase) and metabolism of short-chain alcohols pathway key enzyme gene ADH1(Alcohol dehydrogenase)differentially expressed in the sterile line and maintainer line. In order to study the relationship between CMS and energy metabolism differentially expressed genes, we used CMS P3A and its maintainer line P3B as materials, established the method of isolation of high purity total DNA and RNA, and then cloned the energy metabolism differentially expressed gene6-PGDH and ADH1.We also took semi-quantitative RT-PCR to analyses expression model of6-PGDH and ADH1gene. The main results are shown as follows:(1). Successfully extracted high-quality total DNA and total RNA from kenaf anthers.(2). This research is based on kenaf transcriptome data and the DNA microarray results. The author screened the related differentially expressed gene sequence fragements rigorously which was submited to NCBI by Blast N&Blast P retrieval, and then compared these fragements with6-PGDH and ADH1genes from other species to find the sequence homology. Under the premise of confirmed data reliability, the author obtained the full-length cDNA sequences with bioinformatics methods, and cloned the full-length of6-PGDH gene and ADH1gene by using male sterility (P3A) and maintainer line (P3B) anthers cDNA and total DNA as template. Sequencing analysis showed that the6-PGDH and ADH1gene in kenaf have a high homology with other plants, which indicates that the amplified gene fragment was correct. The full-length of6-PGDH gene is1751bp containing an open reading frame (ORF) of1458bp, the gene of encoded a protein with485amino acids and the6-PGDH gene has no intron. which was amplified by using male sterility (P3A) and maintainer line (P3B) anthers cDNA as template is1300bp, while by using DNA as template, The full-length of ADH1gene is2256bp. The full-length of ADH1containing an open reading frame (ORF) of1071bp, the gene of ADH1encoded a protein with356amino acids and the ADH1gene contains eight exons.(3) Semi-quantitative analysis was performed to6-PGDH and ADH1gene, the result showed that6-PGDH and ADH1gene were down regulated in the CMS (P3A).(4) Successfully constructed6-PGDH specific RNAi silencing gene expression vector.(pART27-pKANNIBAL-R1+2) with a forward and reverse insertion fragment of389bp. This research lays a solid foundation for the further study on kenaf6-PGDH gene function. The research using the method of pollen tube channel genetic transformation is underway.