| China is the world’s largest citrus producer and its citrus planting area and yield both banked first in the world.In recent years,not only the development of citrus canned processing and export raises steadily, but also the orange juice processing industry makes great progress in China.However,there are some technical problems affecting juice quality during the citrus process. The delayed bitterness occuring gradually in juice proceeds from some citruses is one of the obstacles that impedes the development of the industry of orange juice processing.The delayed bitterness is induced by the conversion of limonoate A-ring lactone (LARL) to limonin when fruits getting damaged. LARL is a non-bitter precursor of limonin existing in intact fruit tissue and it is convered to tasteless limonin glucosides, such as limonin17-L-D-glucopyranoside (LG) during the maturation of fruits.Therefore, it is feasible to eliminate the delayed bitterness through preventing the convertion of LARL to limonin by decreasing the accumulation of LARL in mature fruits. It is shown that the citLGT gene from Satsuma mandarin(Citrus unshiu Marc.) encodes the Limonoid UDP-glucosyltransferase enzyme which catalyzes the conversion of LARL to non-bitter LG.Here, the regulation of ectopic expression of citLGT gene was performed through Agrobacterium-mediated genetic transformation of Citrus sinensis (Linn.) Osbeck cv. Jin Cheng.The result of our study offered some transgenic lines with overexpressed and downexpressed citLGT gene for the illuminating the molecular mechanism and the genetic improvement of delayed bitterness in future. The main results are as follows:1. Amplification of citLGT gene and three RNAi functional fragmentsThe coding sequence of citLGT gene was cloned from Satsuma mandarin{Citrus unshiu Marc.) by PCR; Three RNAi functional fragments were also amplified with three pairs of specific primers as the construction strategy and principle of RNAi vetor.The length of three fragments were268bp,176bp and302bp,respectively.2. Construction of plant expression vectors with overexpressing and RNAi-suppressing citLGT driven by CaMV35S promoterThe citLGT and three RNAi fragments were cloned to pMD19-T and PGEM-T, respectively.Then the overexpressing vector named as p35S:citLGT and three RNAi-suppressing vetors named as p35S:citLGT-RNAi-1,p35S:citLGT-RNAi-2and p35S:citLGT-RNAi-3were constructed.In addition,a control vector named as p35S:intron,which contains the intron of chalcone synthase A gene (CHSA) without RNAi fragments,was constructed.3. Agrobacterium-mediated transformation of Jin Cheng and identification of transgenic plantsThe above plant expression vectors were transferred into Citrus sinensis (Linn.) Osbeck cv. Jin Cheng by Agrobacterium-mediated method.GUS histochemical straining and PCR analysis were performed to screen and identify positive transgenics.47postive transgenic plants were obtained in this study.4. Southern blot analysis of transgenes in transgenic plantsGenomic DNA was extracted from transgenic plants. Southern blot of GUS gene was conducted to analyse the integration of foreign genes into genomes of transgenic plants. The results showed that foreign genes were successfully integrated into citrus plant.5. Expression analysis of citLGT gene in transgenic plantsTotal RNA were extracted from leaf tissues of transgenic plants. Using actin gene as a housekeeping control, the expression of citLGT gene in transgenic plants was investigated by Real-time quantitative PCR.PCR results showed that a significant decrease and increase in the expression level of citLGT gene was detected in the RNAi-suppressed lines and overexpressing lines, respectively,compared with the non-transgenic and the p35S:intron transgenic lines. |
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