Studies On The Sucrose Contents Of Fresh Seeds And Functional Analysis Of Key Genes Involved In Sugar Accumulation In Soybean

Vegetable soybean is a specialized soybean (Glycine max (L.) Merrill), harvested in the R6-R7growth stage with full seeds and still green pods. Traits of100-pod weight,100-seed weight and sucrose content are the most important factors of appearance and eating quality for vegetable soybean. Based on the goals of vegetable soybean breeding and genetic improvement, we evaluated323cultivated soybean germplasms in China, and identified quantitative trait loci (QTL) related to100-pod weight,100-seed weight and sucrose content in fresh soybean seeds. The key enzyme genes involved sugar accumulation of vegetable soybean were cloned, then the expression pattern of these genes in soybean tissues were analyzed, together with the determination of sugars contents and enzymes activity. These genes were transformed into wild type and cinvl mutant of Arabidopsis thaliana for functional analysis. Main results are as follows:1. A number of elite germplasms were identified base on the vegetable breeding objectives. A total of58marker-trait associations related to100-pod weight (28),100-seed weight (21) and sucrose content in fresh seeds (9) were identified by association mapping, involving36different SSR markers, the loci alleles which were significantly associated with the traits were analyzed further. Five, four, and two QTLs were detected relating to100-pod weight,100-seed weight and sucrose content in fresh seeds by linkage mapping, respectively. Four SSRs (Sattl36, Satt208, Satt251and Satt445) were found to be related to the pod and/or seed weights traits using the both mapping methods.2. Two soybean cytoplasmic invertase (CInv) genes were homologous cloned and designated GmCInvl and GmCInv2, respectively. GmCInvl contains a2040bp open reading frame (ORF), encoding a680amino acid polypeptide (76.96kD). GmCInv2contains a1665bp ORF, encoding a555amino acid polypeptide (63.30kD). The phylogenetic analysis indicated that GmCInvl and GmCInv2were separated with the acid invertase genes, and all the cytoplasmic invertase genes were clustered together. Two sucrose phosphate synthase genes in soybean were identified, and designated GmSPSl and GmSPS2, respectively. GmSPS1and GmSPS2both contain a3177bp ORF, encoding a680amino acid polypeptide. The phylogenetic analysis indicated that GmSPSl and GmSPS2protein had more closed relationship to Vitis than others.3. The sucrose content in the fresh soybean seeds keeps increasing in the early development stage, and decreased in the later stage, while the glucose content gradually decreased during the whole development stage. The starch content change trends of the developing seeds was different in the three evaluated soybean cultivars. The sucrose content was higher than glucose content in the root, stem, leaf and flower. The activity of cytoplasmic invertase (CInv), vacuolar invertase (VInv) and-cell wall invertase (CWInv) all had a decreased trend during the seed growth. The Inv activity in leaves and flowers was higher than that in stem and root. The activity of sucrose phosphate synthase (SPS) have not a stable change mode in developing seed, and the flower or root usually shows a higher SPS activity. GmCInv1and GmCInv2gene were detected in all different tissues, and expressed strongly in roots. The GmCInv1transcription levels were gradually up-regulated with seed development, which were different from GmCInv2, and GmCInv2is more effective for CInv activity in soybean seeds. GmSPS1and GmSPS2have a similar expression pattern to GmCInv1. The expression level of these genes affected the activity of the corresponding enzymes, and changed the sugar contents, but none of the genes or enzymes could regulate the sucrose or glucose level individually.4. GmCInv1and GmCInv2were transformed into wild type (WT) and cinvl mutant of Arabidopsis, cinvl, CINV1-deficient plants, had reduced activities of neutral invertase (cytoplasmic invertase). Transcripts of the target genes can be detected in transgenic lines except Line11. The GmCInv1and GmCInv2expression in Arabidopsis have higher CInv activity. The sucrose contents and value of sucrose content/glucose content of GmCInvl and GmCInv2transgenic lines were lower than that of controls. The growth and development of these transgenic lines was effected by GmCInv1and GmCInv2, the root length, plant height, rosettes diameter, rosette leaf number, stem number and flowering time were increased in different transgenic lines. The expression of soybean CInv genes complemented the deficient phenotype of the cinvl mutant in some degree. 5. Five of the six GmSPSl and GmSPS2transgenic lines have not significant differences in SPS activity compared with WT, though the transcript of GmSPSl and GmSPS2can be detected in corresponding lines. The significant line Line5has a higher sucrose level than WT, the length of seedlings roots and plant height was increased too. There were no significant SPS activity in other five lines may due to the effect of some certain repressors in the Arabidopsis plant.