Variation Study In Soybean After Introduction Of Trypsin Inhibitor RNAi Expression Vector

Soybean is one of the main sources of plant oil and protein for human beings and animals,and it is cultivated all over the world.However,there are many nutritional inhibitory factors influence the quality of soybean such as soybean Trypsin inhibitor,Lectin,Lipoxygenase,and so on.Soybean Trypsin inhibitor is one of the main inhibitory factors influencing soybean quality and that are not good for human and affect food quality.So it is very meaningful to breed low and null Trypsin inhibitor soybean.It is extremely slow to select and breed species bytraditional methods.With the development of molecular biology,we can use technology of gene engineering to improve.After Rhodes and other scientists got an integrated transgenic plant of soybean in 1988,we have made progress in the establishment of soybean receptors system,researches on transformation techniques and improvement of important trait characters that become more and more important in crops breeding.RNA interference(RNAi) is a phenomenon of special post-transcriptional gene silencing mediated by induced dsRNA with endogenous or exogenous homogenous sequences,a lot of researches have been applied to demonstrate it as a reserved mechanism for gene regulation in animals,plants and fungi.In the past decade, RNAi aimed at composition of crop for nutrition improvement had been developed and tested,which included improving the quality of fat,oils and starch,improving crop quality through altering composition of crop, elevating anti-browning nature,prolonging fruit-store,procuring secondary metabolite by metabolism regulation.This technology,as a knockdown technology,has been applied to a wide range of species to research plant gene function,silence the expression of specific genes for crop genetics breeding and offers alternative approaches for improving crop quality.This research is the prophase's continue & thorough.In the past,the full-length DNA fragment of soybean Kunitz-type trypsin inhibitor gene KSTI3 had been isolated by polymerase chain reaction(PCR),and an RNAi expression system had been constructed.Then the construction had bean introduced into soybean through pollen tube pathway method in order to suppress the expression of soybean trypsin inhibitor.In this paper,the research that at the base of get the transgenic T0 to continue the investigation that the effect about trypsin inhibitor RNAi expression vector to the transgenic T1 quality character and agronomy character.We hope to found some new species that have excellence grace—low or null content trypsin inhibitor—and have good comprehensive characters from some variant progenies.The results of study were acquired as fellow:1.In the next year,the transgenic T0 seeds were growed which had bean introduced the RNAi expression system,when the seeds sprouted,using the genomic DNA from single plant's leaves as template,and PCR amplification was performed with 35S promoter primers.The 830bp PCR product was obtained respectively from nine transgenic plants.While this product disappeared from those control plants that have not been transplanted.Then,PCR-Southern blot analysis was performed to further identify the number of copies that of gene integrated into the geromic DNA of the transgenic plant,And the result indicates that hybrid signal occurred in all the nine plants.No hybrid signals occured in plants that had not been transplanted.And that indicates that introduced genes have been intergrated into the genomic DNA of transgenic soybean.2.Use the transgenic T1 seeds,the expression of Trypsin inhibitor protein was determined by SDS-PAGE analysis.This result indicates that the RNAi technology inhibited KSTI gene expression and lead to the decrease of origin Trypsin inhibitor protein.3.The protein and oil contents in the seeds of the transgenic soybeans were determined by near-infrared spectroscopy with a near-infrared grain analyzer,and the results were compared with those by the Kjeldahl method for protein content and the Soxhlet method for oil content,respectively.The results showed that the tested values were basically consistent across the two different analyzing methods using the same samples. The oil content determined by the near-infrared grain analyzer was more accurate than that for protein content,and the variation was less than 2 and the least variation was only 0.04,indicating that the calibration equation for protein and oil determination is stabile and accurate with the near-infrared spectroscopy.Based on the results,we concluded that near-infrared spectroscopy is a rapid,accurate and nondestructive method for protein and oil determination,and could be used in large-scale screenings of soybean seeds for breeding selection and quality verification.4.Five high content of oil variant were obtained through determining the protein and oil contents in the seeds of the transgenic T1 soybean,one of them come to 27.15%.It shows that only five transgenic soybean effectively integrate the genomic and successfully express siRNA,it leads to the decrease of origin Trypsin inhibitor mRNA,and then the expression of Trypsin inhibitor protein sharply decreased.They are were Jinong 18-32-1-1,Jinong 18-33-1-2,Jinong 18-35-1-1,Meiyinl-2-1-1和Tongnong 14-9-2-1.In the other side,One high content of protein variant was also obtained,the content of protein is 49.89%.5.Nine transgenic soybeans produce Agronomic Character variants at different types and different degrees through plant laboratory observing of the transgenic T1 soybean.The seeds eaten of Jinong 18-32-1-1,Jinong 18-33-1-2,Jinong 18-35-1-1,Meiyinl-2-1-1和Tongnong 14-9-2-1 were increase.Because Trypsin inhibitor have effect on insect-resistanceto the soybean,The seeds eaten were increase further show that the five transgenic soybean effectively integrate the genomic and successfully express siRNA,The expression of Trypsin inhibitor is successfully suppressed with RNAi.and then the transgenic soybean produce genetic variants.