The Studies On Construction And Genetic Transformation Of RNAi Plant Expression Vector Carrying Cytochrome P450from Castor

In process of plant cell division and embryonic development, plant hormonebrassinosteroid （BR） and gibberellin （GA） involved in the development of internodesinfluence the plant height. Deletion of BR and GA will show the dwarf phenotype. TheSynthetic and Regulation of hormone BR and GA were affected by many enzymes, most ofwhich belong to the family of cytochrome P450, whose genes have a significant impact on thedevelopment of plant stem. Homology of amino acid sequence was60.98%compared withCytochrome P450from castor and rice, which showed two genes belong to the samesubfamily, and we can speculate that the function of two genes was similar. Two genefragments have been cloned and RNAi expression vector have been constructed successfullyaccording to the nucleotide sequence of cytochrome P450genes from castor which have beenpublished in order to verify functions of cytochrome P450gene of castor and reveal therelationship between the gene and hormone synthesis. Effects of different types andconcentrations cytokinin and auxin on regeneration shoot and root of explants were studiedusing cotyledons, hypocotyls and cotyledonary node of “Tongbi5” as explants, and higherfrequent regeneration vitro system of cotyledonary node of castor was obtained. Transgeniccastor plant was gotten by co-culture of cotyledonary node and Agrobacterium, which hasprovided the foundation for study functions of Cytochrome P450of Castor. The main resultswere as the follows:1. Two pairs of primers were designed basing on nucleotide sequence of cytochromeP450of castor which was published by Genbank（XM₀02525863.1）, and two fragments ofP450gene named as P450s and P450a were gotten by PCR, and sequence and comparisonwas carried out after connecting successfully with the cloning vector.The results showed thatlengths of P450s and P450a were523bp and411bp and had98.28%and99.51%sequencesimilar with the original respectively. These results indicated the cloned fragment wascytochrome P450of castor.2. Recombinant plasmid pMD-P450s and intermedium vector pHANNIBAL weredouble digest by SacI and KpnI and inserted the P450s into Linear intermediate vectorpHANNIBAL, and vector pHAN-P450s was constructed, which was correct by double digestion and PCR. Then plasmid pMD-P450a and vector pHAN-P450s were recombined bydouble digest by XbaI and Handâ…¢ and inserted the P450a into linear intermediate vectorpHAN-P450s, and pHAN-P450s-P450a vector was constructed, which was correct byenzymes digestion and PCR. At last, recombinant plasmid pHAN-P450s-P450a and plantexpression vector pBI121were double digest by SacI and XbaI and pHAN-P450s-P450a wasinserted into linear intermediate vector pBI121, and RNAi plant expression vectorpBI-P450-RNAi was constructed, which was correct by enzymes digestion and PCR. Theplant expression vector pBI-P450-RNAi was successfully inserted into the AgrobacteriumLBA4404, which was correct by enzymes digestion and PCR, Agrobacterium culture mediumfor infection was successfully gained.3. Regeneration system of “Tongbi5” was established: after analyzing regenerations ofshoot, root from cotyledons, hypocotyls and cotyledonary node and transplanting survival rateof regeneration plantlets in general, the result was as follow: the optimum regenerationexplants of “Tongbi5” was cotyledonary node and the optimum cutting time was beforecotyledon head emerging from endosperm when radicle and hypocotyl was3-5cm.Cotyledonary node that showed “” after cutting two-thirds of cotyledonary and all radicle,divided it into two parts and inserted into the medium vertically. And the optimum mediumwas1/8MS+8.0mg/L ZT, which regeneration frequency of explants achieved for75.6%andaverage regeneration buds per explants was2.6. The optimum medium for root differentiationwas1/8MS+2.0mg/L NAA, which root regeneration frequency achieved for86.7%andmultistory stocky taproot with many branch roots could be differentiated and hadhigh-survival rate after transplant.4. The genetic transformation system of castor cotyledonary node was established:cotyledonary node of “Tongbi.5” was cut by patterns of vitro culture, pre-cultured onmedium（1/8MS+20mg/L AS+8.0mg/L ZT） for2-3days, infested10minute withagrobacterium culture medium LBA4404with the concentration of OD600=0.8, thencotyledonary node was co-cultured for3days after the remnants of bacteria was absorbed bysterile filtered paper, transferred cotyledonary node into the resistant screeningmedium（1/8MS+250mg/L Kan+300mg/LCef）, it took40days for shoot differentiation and root regeneration. Regeneration plantlets was transplanted and detected by PCR and Southernblot, and proved it was positive transgenic plants.5. P450gene of transgenic castor interfered by RNA was inhibited through Northernblot. One band of peroxidase and esterase isozyme in transgenic castor leaves was less thanthat of non-transgenic castor. Phenotype characteristic of the transgenic castor were follow:plant height was dwarfed, leaves were smaller, leaf color was dark green. These entirepreliminary proved that the gene of P450was closely related to plant height of castor.Western blot analysis showed that the expression of P450protein of transgenic castordecreased significantly compared with the control...