Cultivation Of Marker-Free Transgenic Rice Of Lysine-Rich Protein Gene

Rice is a main food crop, but its lysine content is low and is the first limiting amino acid, which severely limits the absorption and utilization of other amino acids. The nutritional value of rice can be increased greatly by improving the content of lysine in the seeds and the utilization of the entire protein. Therefore, it is of important significance of cultivating lysine-rich rice. The development of transgenic technology and discovery of new lysine-rich protein gene make it possible of cultivating excellent lysine-rich rice varieties. At the same time, with the commercialization of transgenic plants, the safety issues are becoming increasingly aroused people’s attention, among which marker genes is the main problems.On the basis of the problems above, in this research the high lysine genes which came from soybean seeds were transferred into rice using the methods of Agrobacterium mediated genetic transformation and double T-DNA vector technology to cultivate marker-free transgenic Lysine-rich rice.The main research results were summarized below.1. Optimization of rice transformation systemRice varieties, maturity of rice seed, sterilization methods, cultures and conditions of callus inducing, rice callus status used for infection and screening pressure in differentiation stage were all optimized in this research, and the results showed that three rice varieties (TY302, U131, HJ19) can be used as preferable materials in rice genetic transformation, and newly harvested rice seeds were more preferable than the aged ones as the material for callus induction, and25%NaCIO20min+0.1%HgCl2lOmin was favorable as the sterilization condition, also the calli induced under light were better than those in the darkness, meanwhile the rice calli after3-5times subculture were better in differentiation than the newly induced used for infection, finally the appropriate concentration of Bialaphos was0.8mg/L for rice calli during differentiation stage.2. Genetic transformation and obtain of the To transgenic plantsFive plant express vectors (pTTBUG2, pTTBUG8, pTTBGG2, pTTBGG8, pTTBUP2G2) were transfered into rice varieties (TY302, U131, HJ19) using the method of Agrobacterium mediated genetic transformation, and.149regenerated plantlets were obtained 3. Screening and detection of the To regenerated plantletsBasta resistance of the To regenerated plantlets was tested. All of the149regenerated plantlets were smeared50mg/L of Basta in the leaves during seeding stage, among which124plantlets showed significant resistance to Basta, and88of the resistant plantlets were verified transgenic plants harboring bar gene, furthermore,46plantlets were confirmed harboring Lysine-rice protein Gene after PCR detection. The positive rate and co-transformation rate respectively were70.1%and52.3%.4. Amino acid composition analysis of To transgenic Lysine-rice rice seedsThe amino acid composition and content of the To seeds of transgenic plants was measured. The results show that, Its amino acid composition and content was no significant increased in the six transgenic, In the lysine percentage of the total amino acids, Only transformed plants HJ19*P2G2-23lysine was increased by5.14%5. Screening and detection of the T1transgenic plantletsTo transgenic seeds were inoculated on1/2MS culture containing5.0mg/L Bialaphos and cultivated under30℃/light, after one week, parts of the seeds can not grow into plantlets then gradually dead, on the contrary, other To transgenic seeds showed significant Bialaphos resistance and grew normally. Genetically modified rice strain TY302*U2-36To seed was cultivation to the greenhouse, PCR detection of the T1transgenic rice plantlets was conducted, and the results showed that6plantlets contained GsHLP2gene whereas without bar gene among the16detected plantlets. The6plantlets contained GsHLP2gene plants was detectioned by RT-PCR, results were positived.