Function Analysis Of Sulforaphane Accumulation Relative Gene MY And SMT In Broccoli

Broccoli(Brassica oleracea L. var. italica planck) is one of the most important import vegetables in developed countries. It has high value nutrition and rich in Selenide. Anymore, it is also rich in sulforaphane which is the highest active substance for anti-cancer in vegetables. Sulforaphane is synthesized by myrosinase (MY) hydrolyzed glucoraphanin. Broccoli sulforaphane content would be decreased rapidly while there is higher selenium fertilizer. Selenocysteine methyltransferase (SMT) transferred selenide into nonprotein anmio acid with Selenium and stored in plant. It maybe relate with regulation of sulforaphane. In this study, function analysis of MY gene and SMT gene is carried out. This study would provide a theory evidence for sulforaphane regulation and high sulforaphane broccoli variety breeding. The main results are as follow:(1) The over-expression and RNAi expression vectors of MY gene(was cloned from chinese broccoli) was constructed and transformated into broccoli, and empty vector (pCAMBIA1301) as control. PCR analysis results showed that 64, 49 and 47 were positive transformated plant for vectors, respectively. The positive percentage was 90.14%, 85.96% and 88.68% respectively. GUS expression showed that 81.69%, 77.19% and 75.47% of transformated plants be stained into blue. Furthermore, southern blot analysis showed that the aimed fragments from 3 vectors were inserted into broccoli genome by single copy.(2) The relative expression of MY gene in transgenic broccoli was analysis by real-time PCR. The relative expression of MY gene was significantly increased 16.5% in over-expression plants and decreased 23.8% in RNAi plants in average. Sulforaphane content was increased 92.77% in MY gene over-expression broccoli head and decreased 35.82% in RNAi expression. The results showed that MY gene from chinese broccoli should participated in sulforaphane synthesis.(3) The over-expression and RNAi expression vectors of SMT gene(was cloned from broccoli) was constructed and transformated into broccoli, and empty vector (pCAMBIA1301) as control. 60, 67 and 47 positive transformated broccoli from 1301-SMT, 1301-SMT RNAi and pCAMBIA1301 were obtained, respectively. The positive percentage was 88.24%, 91.78% and 88.68% respectively. GUS expression showed that 82.35%, 80.82% and 75.47% of transformated plants be stained into blue. Furthermore, southern blot analysis showed that the aimed fragments from 3 vectors were inserted into broccoli genome by single copy.(4) Real-time PCR experiment showed that the relative expression of SMT gene was significantly increased 13.93% for over-expression plants and decreased 15.6% for RNAi in average, comparation to non-transgenic broccoli. Sulforaphane content in transgenic broccoli head was increased 64.12% by over-expression of SMT gene and no significant changed by RNAi expression of SMT gene. After Na2SeO4(5.2 mmol/L) spraied, sulforaphane content was decreased 14.09% in SMT gene over-expression broccoli head compared to control. No sulforaphane found in SMT gene RNAi expression broccoli after Na2SeO4 sprayed. The SMT gene should plays an important role in sulforaphane accumulation, especially in relieving the decrease of sulforaphane caused by Selenium fertilizer stress.