Simultaneous Improvement Of Protein Quality And Pest-resistance In Rice Using Sb401 And Pta Genes

Rice (Oryza sativa L.) is one of the most important food crops in the world, and it is also staple food of more than fifty percent population in China. But the damage resulting from rice pests is disastrous sometimes. According to statistics, the losses of yield will be eighty percent if chemical pesticides are not used. Planthopper is one of the most serious rice pests, so controlling planthopper is an important pathway to improve rice yield. Although chemical pesticides can decrease the planthopper losses to some extent, it will result in new troubles such as environmental pollution and health harm. With advent and development of gene engineering techniques, new thinking is obtained to control rice pests including planthopper.As staple food of majority, high and stable yield of rice is first paid more attention by people. With the living standard being improved, people more and more focus on the nutritional value of rice especially the content of protein and lysine. However only 5 to 10 percent of grain weight in rice seeds is protein and only 3.5 to 4.0 percent of protein is lysine which is the first limited amino acid in rice. Absence of lysine will bring about disturbance of body metabolism even some diseases relating to physiological function, and therefore show the content of lysine is closely bound up with the persons who live on rice. So raising lysine content to improve protein traits is an important target of rice breeding.We can improve multiple traits of rice such as resistance to pests and protein quality at the same time by conventional breeding approaches or sexual crossing between transgenic plants, but they both cause long breeding cycle and low efficient. Introducing multiple genes which present in the same T-DNA region of one plasmid into target rice is an efficient approach to improve multiple traits of rice simultaneously. In this study, pinellia tenata agglutinin (pta) gene which are driven by a maize ubiquitin gene promoter (Ubi) and a lysine-rich protein (sb401) gene which are controlled by a rice endosperm-specific expression (PGlu) promoter were transferred into two rice varieties via Agrobactehum-mediated genetic transformation. The two genes were carried in the same T-DNA region of plasmid pDB13PS. The results were as follows:1. After the embryogenic calli derived from immature embryo of japonica rice Nipponbare and indica rice 501R co-cultivated with Agrobacterium tumefaciens strain EHA105/pDB13PS for 2-3d and selected for three times on selection medium containing 25-45 mg/L hygromycin, 155 resistant calli (ninety Nipponbare and sixty-five 501Rrespectively) were achieved from 527 test calli. The resistant rates of the two varieties were respectively 63.38% and 16.88%. Eighteen positive transgenic plants (thirteen Nipponbare and five 501R respectively) were identified by PCR, which resulted in transformation rates of 9.15% and 1.30%.2. Total DNA of two transgenic plants identified by PCR and one non-transgenic plant was extracted to perform southern blotting assay. DNA hybridization results showed that the two transgenic plants have 1 to 2 hybridization bands, but the non-transgenic plant has no hybridization signals. It indicated that the sb401 and pta genes have been successful introduced and co-integrated into the genome of the receptors.3. Total RNA was isolated from leaf tissues of three transgenic plants (two identified by PCR and one analyzed by southern blot) and one non-transgenic plant to conduct RT-PCR analysis for the pta gene. RT-PCR analysis showed that the three positive transgenic plants but the negative control had the expected size bands (810bp). It demonstrated that the pta gene has expressed at transcription level.4. 100- 150 rice seeds were randomly picked out from each of the 10 transgenic rice plants to determine the content of lysine and total protein. 50 seeds from each of four non-transgenic plants were selected and mixed as the negative control. The lysine and total protein content in the seeds, compared with negative control, was also improved differently in transgenic rice plants. The results suggested that the sb401 gene has effectively expressed in rice seeds.5. 60 seeds of each To generation transgenic plants which have been measured in total protein and lysine content were selected and divided into 3 groups to test resistance to hygromycin B. The seeds of non-transgenic plants were used as negative control. The data showed that the ratio of resistance to sensitivity is three to one which follow Mendel's dominant inheritance law of single gene.