RNAi Vector Construction Of Ricin Family And The Transformation Of Low-toxicity Castor(Ricinus Communis L.)

Ricin is a deadly natural toxin, mainly found in the castor seeds, and it can cause shock or death of all mammals by excessive intake. The de-oiled castor bean cake contains various nutrients and high amounts of proteins, but it could not be directly used in animal husbandry production due to the ricin in it. Therefore, detoxification treatment is necessary for its application, but results in loss of nutrients and a waste of resources. To obtain the low-toxin castor lines, the RNAi technology which can block the synthesis of ricin will be performed to provide a very important and feasible way for comprehensive utilization of the castor bean cake and proteins. In this study, we first analyzed the temporal and spatial expression patterns of the RICIN precursor gene and found out23members in the RICIN gene family, then clustered them into7sub-groups. A fragent of highly conserved region was selected from each sub-group. Finally, the7selected fragments were ligated together to construct a combined RNA interference vector and to be transformed into castor. The main results were as follows:1. According to the sequence of RICIN precursor gene （XM₀02534603.1） deposited in Genebank, all the homologues were found and analyzed. There were22sequences which have fully open reading frame to be selected and analyzed by BLAST （basic local alignment search tool）. Based on the highly conserved region from the alignment, primer pairs were designed to detect the expression profile of RICIN precursor gene in caster.2. The expression profile analysis of the RICIN precursor gene during the development of caster fruit.Though the semi-quantitative RT-PCR and real-time quantitative PCR, we found that the RICIN precursor gene did not express in any examined tissues at the seeding stage and only in the roots and seeds at the flowering stage. During the fruit development, the RICIN precursor gene was expressed in ovaries within first3days after pollination （DAP）, and then diminished. Until at24DAP, the expression of RICIN precursor gene was observed and was rapidly increased.3. The23members of ricin gene family were named from RC-1to RC-23and classified into7sub-groups by clustering analysis:sub-groupl（RC-2,4,5,7,11）, sub-group2（RC-1,6,9,10,12）, sub-group3（RC-3,8and13）, sub-group4（RC-15and RC-23）, sub-group5（RC-18,19,20,21and22）, sub-group6（RC-14and RC-17）, sub-group7（RC-16）.4. We cloned the highly conservative sequence of each sub-groups as follows: sub-groupl （400bp）, sub-group2（182bp）, sub-group3（100bp）, sub-group4（185bp）, sub-group5（143bp）, sub-group6（126bp）, and sub-group7（324bp）, then ligated them together, named RICIN. This combined fragment RICIN was inserted into pFGC1008plasmid in forward and reverse direction to construct the RNAi vector which was subsequently used to interfere with the ricin gene family in vivo.5. By the means of genetic transformation system for Ricinus communis:embryo axes pre-cultured for5days under dark conditions were excised. Then they were immersed in bacterial suspension （OD6oo=0.2-0.5） for10mins.Subsequently, the explants were co-cultivated for3days. Following co-cultivated, the explants were transferred to the resitant screening medium containing20mg/L Hyg. The whole process takes about75days. Reisitant seeding were proved to be positive transgenic plants by PCR. Results of genetic transformation:we obtained97resistant seedings in total and one transgenic plant, the rate is6.93%and0.07%respectively.