Cloning, Function And Molecular Evolution Of The Key Genes Of Strigolactone Synthetic Pathway In Soybean

Strigolactones are newly discovered plant hormones that perform various functions from signaling in symbiotic interactions with arbuscular mycorrhizal fungi to controlling outgrowth of axillary buds. Some target genes of the pathway of strigolactone biosynthesis have been cloned and studied in Arabidopsis, pea, rice and other plants. The number and distribution of branches are important traits of plant type, and also influence seed yield in soybean. However, the branch traits are low heritability and vary due to different environmental factors, so little information of the genes for branches is available in soybean. The released whole-genome sequence of soybean can be utilized to reveal the function and molecular evolution of the target gene (protein family), and the overall understanding of the gene characters can be obtained through combining the methodologies of molecular biology with evolutionary analysis. In this thesis, gene diversity, function and molecular evolution of the important genes in strigolactone synthetic pathway were analyzed to present useful information for gene mining and molecular breeding in soybean. The main results are as follows.(1) GmMAXl, a key gene of strigolactone synthetic pathway, was cloned from cultivated soybean Kefeng 1 Hao through RT-PCR technique. Its sequence length is 2709 bp, including 4 exons and 3 introns. The length of ORF is 1362 bp coding 454 amnio-acid. GmMAXl belongs to CYP711A of P450 super-family. It expressed not only at roots, shoots and leaves, but also at reproductive tissue and even at calli, indicating the correlation between the gene and the development of plant architecture as well as reproductive organs. The transgenic Arabidopsis plants performed less branch number and longer growth period than the wild types.Total 19 Glycine max and 18 G. soja accessions were selected to resequence GmMAXl gene. Thirty polymorphic nucleotide loci including 22 SNP and eight INDEL were detected among the tested materials. Three loci cause the change of amino acid. There were fifteen haplotypes in these soybean accessions, and thirteen in G. soja and six in G. max. The H1 and H3 haplotypes were predominant in G. max. The Tajima'sπvalues were 0.00785 and 0.00226 in G. soja and G max respectively, indicating the conspicuous diversity in wild soybean.(2) Carotenoid cleavage dioxygenase genes CCD7 and CCD8 are involved in upstream consecutive steps of the proposed strigolactone biosynthesis pathway. Total nine candidate CCD genes in soybean whole genome were identified according to the published Arabidopsis thaliana CCD protein information. These genes distribute on eight chromosomes, and can be divided into NCED, CCD1, CCD7 and CCD8 clades. Each clade had similar motifs and gene structure. The CCD members experienced different evolution fates. It presumed that Gm12g36530 and Gm08g10190 might be the major genes corresponding to the synthesis of the carotenoids and abscisic acid. The NCED has been divided into two clade, one was related to ABA and another without identify function.The CCD7 and CCD8 sequences from eleven model species were further analyzed to reveal their phylogenetic relationships. It showed that these species can be divided into two clades corresponding to sequences from monocotyledons and dicotyledons but the sequences from the primitive moss, Physcomitrella patens, appeared to be evolutionarily distinct from the angiosperm RMS. CCD7 and CCD8 are rather conservative. As essential genes in the strigolactone pathway, the divergence timing of the conserved CCD7 and CCD8 genes reflect the approximate generation time of strigolactone as a regulatory substance. The results of Ks values indicated that CCD7 and CCD8 diverged about 290 to 430 million years ago with an average of 360 million years. The timing calculation was in accord with the initiative time of symbiosis between plants and microorganisms inferred from the fossil record.