Soybean Lipoxygenase Gene Rnai Expression Vector Construction And Expression Regulation

The purpose of this research is to decrease the content of lipoxidase in soybean and increasethe content of soybean oil, hope to found some new species that have excellence grace. Withgenetic engineering method, the lipoxidase gene of core consensus sequence were cloned, ahighly efficient with hpRNA and ihpRNA of RNAi expression system were constructed. Thenthe gene constructs were introduced into soybean mediated by pollen tube passage method inorder to restrain the gene expression of lipoxidase and regulate the biological composing processof lipoxidase. The main results are as fellow:1. Transform the plant expression vector pCAMBIA1301, removal the hygromycin, and add35s promoter according to anti-clocwise from plasmid pBI121.2. Using the genomic DNA of soybean variety "JiNong 18" as template, to select the highesthomology from the three lipoxidase of isozyme. The lipoxidase was isolated by PCR and wascloned into pMD18-T Vector. The size of this promoter is 357bp. Sequencing analysis of thislipoxidase gene through NCBI GENE BANK showed that was the same to the enunciatedbefore. Using the fragment of soybean lipoxidase gene into pCAMBIA1301 35S promoter, thefragment is norientation and inversus, then, pC1301LoxRi, the hpRNAi expression vector ofsoybean lipoxidase gene was constructed. The To seeds of soybean variety "JiNong18" wasreapped by pollen tube passage method. After the seeds were germinated, using geromic DNA ofthe regenerated plants' leaves as template, PCR amplification was performed withpCAMBIA1301 gus gene primers. The 721bp PCR product was obtained respectively from the12 transgenic plants. Link the extraction of the product to pMD18-Tvector, sequencing analysisshowed the fragment amplified was the gus gene we need. Then, Southern blot analysis wasperformed to further identify the number of copies that of gene integrated into the geromic DNAof the transgenic plant, And the result indicates that hybrid signal occurred in both the transgenicplant lines with one or two copies, respectively. But there was no hybrid signals shown in plantsthat hadn't been transplanted. Using the cDNA from the transgene of soybean as template forRT-PCR, the content of the lipoxidase mRNA in transgene plants decreased obviously.3. Clon a piece of intervening sequence from soybean variety "JiNongl8", the size of thispromoter is 230 bp. The fragment of soybean lipoxidase gene into pC1301LoxRi expressionvector is between the the norientation and inversus. Then the hpRNAi expression vector ofsoybean lipoxidase gene pC1301LoxiRi was constructed. The T₀ seeds of soybean variety "JiNong18" was reapped by pollen tube passage method. After the seeds were germinated, usinggeromic DNA of the regenerated plants' leaves as template, PCR amplification was performedwith pCAMBIA1301 gus gene primers. The 721bp PCR product was obtained respectively fromthe 14 transgenic plants. Link the extraction of the product to pMD18-Tvector, sequencinganalysis showed the fragment amplified was the gus gene we need. Then, Southern blot analysiswas performed to further identify the number of copies that of gene integrated into the geromicDNA of the transgenic plant, And the result indicates that hybrid signal occurred in both thetransgenic plant lines with one or two copies, respectively. But there was no hybrid signalsshown in plants that hadn't been transplanted. Using the cDNA from the transgene of soybean astemplate for fRT-PCR, the content of the lipoxidase mRNA in transgene plants decreasedobviously.4. From the generation transgenic T₁ seeds, the activity of lipoxidase was assayed using theuntraviolet spectrophotometry method. The result indicates that the activity of soybeanlipoxidase which transformed by pC1301LoxRi plasmid decreased by 62%, and the activity ofsoybean lipoxidase which transformed by pC1301LoxiRi plasmid decreased 76%. And theactivity indicates that the insert of introduced has efficiently restrained the expression oflipoxidase.5.The content of lipoxidase also assayed from T₁ generation transgenic soybean usingSDS-PAGE method to test. The result indicate that compare to control, the content of lipoxidaseof transgenic soybean were decreased, among the total which transformed by pC1301LoxRiplasmid average decreased by 55%, and the activity of soybean lipoxidase by pC1301LoxiRiplasmid average decreased 71%.6. Use the transgenic T1 seeds, The protein and oil contents in the seeds of the transgenic soybeans weredetermined by the Kjeldahl method for protein content and the SoxMet method for oil content, respectively.The results showed that the content of protein of transgenic soybean were decreased, among the total,average content of protein which transformed by pC1301LoxRi plasmid were 36.06%,and thenon-transgenic decrease 0.95 persent, the maximum decrease 1.28 persent which to achieve35.73%; the average content of oil which transformed by pC1301LoxiRi plasmid were 23.89%,and the non-transgenic increase 0.74 percent, the maximum increase 1.09 percent which toachieve 24.24%; the average content of protein which transformed by pC1301LoxiRi plasmidwere 35.63%, and the non-transgenic decrease 1.38 percent, the maximum decrease 1.62 percentwhich to achieve 35.39%; the average content of oil which transformed by pC1301LoxiRiplasmid were 24.33%, and the non-transgenic increase 1.18 percent, the maximum increase1.61 percent which to achieve 24.76%. 7. The result shows that the agronomy character of the transgenic T1 soybean has no obviousdifference with the control through plant laboratory observing which include plant height, thenode number, pod numbers, grain number of single plant, the seeds eaten, 100-grain weight.8. The transgenic T₂ seeds were determined by PCR and PCR-Southern blot, the result showsthat the foreign gene may be heridable over successive generations, the segregation ratio wasbasically corresponds with the Mendelian genetic law.9. Construct the RNA components of plant gene applied recombinant PCR was discussed, itpresents preliminary proof that it is a simple, rapid, feasible and wide application range method.10. Indoor conversion condition of pollen tube passage was discussed, it presents that it cancarry out convert soybean indoor by pollen tube passage in the northern part of the country,adding generation one time every year.The study has obtained the good effect and obtained the transgenic soybean whichlipoxidase content obviously decrease and the fat content obviously increase through applyingfor the RNA interference technology to the improvement soybean lipoxidase content at the firsttime, it broked through the shortcoming of the traditional breeding method in the improvementsoybean quality aspect easily the idioplasm resourced limit and the breeding time long, itexplored the new way for improvement soybean quality and enhanced the soybean oil contentusing the RNA inference technology, it has realized the innovation of the soybean qualityimprovement breeding and the high oil breeding method, also has the important theory and thepractice significance. For later through the RNA interference technology improvement soybeanother nutrition inhibiting factors, and lay the foundation for enhance the soybean quality.