Construction Of Technical System For Soybean Germplasm Enhancement Based On Rice Full-length CDNA

Crop germplasm enhancement is the key factor for effective utilization of germplasm resource and the basis for breeding project. During the past 50 years, crop germplasm enhancement had played important role on agricultural cultivars renewal, crop yield increasing and food security etc. Common soybean is the main source of vegetable oil and protein for human being, but its narrow genetic background and longer cycle for germplasm enhancement was difficult to meet the requirement of soybean breeding. In addition, gene patent protection affected the progress of soybean transgenic breeding in our country. So, developing a new type of technique on soybean germplasm enhancement is important to increase efficiency of soybean enhancement and meet the need of soybean breeding.In this study, based on transgenic technique, a new type of soybean germplasm enhancement technique was developed with synthesis of rice full-length cDNA, construction of effective expression vector and optimization of Agrobacterium-mediated transformation system. Examining results of his-chemical and molecular biology for transformed soybean plantlets showed that this technique system was feasible on soybean germplasm enhancement. So, this technique developed provided us a brand-new approach to break through gene patent protection and made excellent agronomic trait genes exchange among different species and generas. The main research results were showed as following:1. Obtaining high quality rice full-length cDNA. In this study, total RNA of rice varieties Jingjing7, Hexi15, Zhenzhu42, liaoyan16, Handao277 and Handao502 expressed in different period were isolated after treatment of drought, salt and cold stress, respectively. Rice full-length double strand cDNAs were reverse transcripted from mRNA by SMART technique and were cloned by pGEM-T easy vector. PCR results indicated that the size of insert cDNA fragments ranged from 0.3kb to 2kb with average size of 900bp.2. Construction of powerful expression vector with different size of rice full-length cDNA. Rice full-length cDNA were ligated to binary expression vectors through blunt-ended and T-A ligation methods respectively, and then were transformed into Agrobacterium tumeflciens which was ready for soybean gene transformation. Detect results showed that these two ligation strategies were feasible with 60%～75% ligation ratio, although the ligation efficiency was low.3. Construction of Agwbacterium-mediated soybean transformation system. A new type of explant was explored for the first time, which was composed of one embryonic tip and one cotyledon of soybean using 3 cultivars of Zhongpin 661, Kennong 18 and Lv75. The regeneration efficiency was more than 90% with Zhongpin661 95%, Kennong18 93% and Lv75 90%, which was higher than that of cotyledonary-node (50%) and embryonic tip (70%). The results showed that the transformation efficiency of Kennong 18 was 10%, and indicated that this new transformation system was a powerful technical platform for soybean genomic study in Agrobacterium -mediated transformation and construction of mutants libraries. 4. Examination of new type of germplasm enhancement: different rice genes ligated to vector were transferred into soybean by primarily constructed Agribacterium tumeficiens-mediated transformation system and more than 500 transformed regenerated plantlets were obtained. The results of histochemical (GUS) and molecular biology (PCR, Southern, etc.) indicated that rice genes had been transferred into soybean genome and obtained rice gene integrated soybean plantlets. Thus the germplasm enhancement technical system based on rice full-length cDNA was feasible.5. Compared with traditional germplasm enhancement, this innovation technical system based on rice full-length cDNA had its advantages. (1) no gene patent restriction: genes used in this technique were came from rice full-length cDNA directly and no need to know their sequence;(2) breaking through reproductive interspecific segregation furthest: this technique made germplasm enhancement could not only happen between different familia and races but also between animal and plant, which would broaden application domain of excellent genes; (3) increasing efficiency of germplasm enhancement: compared with previous plant transgenic method with one gene transformation every transgenic event, this technical system transformed rice cDNA "gene pool", and implemented high throughput gene transfer. At the same time, this system no needed to do time-consuming gene cloning.