Study On Inheritance Expression And Km Resistant Screening Of The C-terminal Fragment Of Arabidopsis CRY1 Gene Transferred Into B.napus L.

A big number of rapeseeds were abtained through floral-dip transformation in B. napus L. and B.campestrisL. after 2003 in the Rapeseed Research Center of Sichan Agriculture University. In this paper, the rapeseeds were studied on inheritance, expression identifying and Km-risistant screening. The main results obtained include:1.The T₂ generation of the transgenic T₁ progeny in B.napus L. cv. westar, which was obtained by floral-dip method, was studied with Km test and the inheritance of the C-terminal fragment of Arabidopsis CRY1 gene was analysed. The positive plants from Km test were examinated with PCR detection and the specific DNA fragment was isolated, sequenced and compared to the original target gene sequence. GUS examination and anthocyanin accumulation in the PCR-positive plants were further examinated to study the experssion of the target gene in thetransgenic plants. The results indicated that the c-terminal fragment of Arabidopsis CRY1 gene was successfully integrated into the rape genome, mainly in the form of tabling, with a few in the form of single copy. The transformed gene could be stablytransmitted in the progeny, with a transmitted line rate of 62.5% . The genetic pattern of the transformed gene in the T₂ generation was mostly non-Mendelian. The transgene was expressed in some of the transgenic plants, but kept silent, in other plants.2.The T₃ generation of the transgenic T₂ progeny in B.napus L. was tested with a NIRS instrument to examine the quality character such as oil content, erucic acid content, sulfur glucoside content. The result showed that, the quality character such as oil content, erucic acid content, sulfur glucoside content of transgetic rape seed and comparison material have not remarkable difference.3.The To generation in B.campestris was studied with three methods such as Km daubing vane, dipping seeds and MS culture medium containing Km to screen T₀ seeds of transgenic, rapeseed tagging NPT-â…¡. The results indicated that the best Km choice press was 200mg/L. The method of Km daubing vane can distinguish to Km-resistance or not for 4-5days.4 plants were chosed in this method and its PCR detection was positive. The reliability reached 100%. The method of dipping seeds can distinguish to Km-resistance or not for 2-3 days. 5 plants were chosed in this method and its PCR was positive. The reliability reached 100%. The method of Km MS culture medium can distinguish to Km- resistance or not commonly for 10-15 days. 20 plants were chosed in this method but only 5 saved in thransplanting. The result of PCR detection is that 3 plants were positive and 2 were negative. The reliability reached 60%. So, it could be thougnt Km daubing vane and dipping seeds were ideal methods for fast screening transgenic rapeseed and descendant's heredity analysis.