Production And Study On New Bt-Transgenic Rice

Rice is one of the world's major food crops which is feeding-more than 50%of the world's population as staple food.The destructive pest of rice is one of the major threat to rice production causing yiled reduction.There are a wide variety of ice pests,mainly including rice borers,rice planthoppers,rice elephants and rice gall midge.These pests cause serious losses to rice production.Presently,using chemical insecticides is the main solution against to rice pests,but it results a series of problems such as environmental pollution,decreased food security and raised production cost.The most effective way to control rice pests is to obtain insect-resistant rice varieties through breeding methods.Among,the transgenic rice is one of the effective methods to obtain insect-resistant rice varieties.Bacillus thuringiensis(Bt)can encode insecticidal active protein genes with high specificity,which are Bt genes.Therefore,breeding of insect resistant cultivars using transgenic technology to transform Bt into rice varieties.In this study,we transferred three codon-optimized exogenous Bt genes(Cry2Ag1,Cry30Gal and Cry54Abl)into rice restorer line Shu-818(R818)by Agrobacterium media-ted method,and we obtained a series of positive transgene plants in early generation.The main results are showing as follows:1.The Cry2Ag1,Cry30Gal and Cry54Abl gene was as optimized based on rice preference codon.The three sequences of the modified genes were removed from the related ATTTA sequence,intron cleavage sequence and PPSS sequence,whilst increased the number of genes in the restriction enzyme cleavage site in the coding region.Compared with the original version of those genes,the G+C contents were increased,and the nucleotide sequence is greater than 84.0%regarding to original version,while the similarity of amino acid sequence is 100%.2.The modified Bt gene have been finally cloning into pDTMAR-Hyg vector to make expression binary vectors pDTMAR-Cry2Ag1-Hyg,pDTMAR-Cry30Gal-Hyg and pDTMAR-Cry54Ab1-Hyg by the method of artificial synthesis.All of positive plasmids were verified by enzyme digestion and sequencing.3.We transformed exogenous genes Cry2Ag1?Cry30Gal and Cy54Abl into caulls of rice restorers R818 by Agrobacterium tumefaciens-mediated transformation approach,and 146,219,164 T0 transgenic plants were obtained.Then we test gentic plants by PCR and Hyg B solution,which only 45,71,40 plants have been successfully transformed Cry2Ag1?Cry30Gal and Cry54Ab1 based on the results of PCR and Hyg B solution testing.The rate of co-transformation were 30.82%.32?42%.24?39%.4.The seeds in T0 generation were haves by mixed collection and growed 20 plants per plot.T1 transgenic plants were detected by hygromycin solution immersion and PCR detection.The results showed that 48,98 and 59 strains of Cry2Agl,Cry30Gal and Cry54Abl were obtained from T1 generation carring the target gene without Hyg marker gene,The efficiency of the selection of marker genes in T1 generation plants was 5.33%,6.90%and 7.38%.We previously created an insect resistant cultivars by transforming a Bt gene named Cry30Fal in rice.In the application of rice varieties,rice cadmium content of heavy metals is considered to be an important food safety index.Whether the Bt transgenic rice is largely unknown.Transfer of heavy metal in is very rare compared with.In this study,the effects of cadmium stress on the growth of Bt transgenic rice were studied in order to evaluate the response of transgenic Bt transgenic rice to cadmium stress.At the same time,the transgenic rice was used in soil cadmium pollution to make a security evaluation.The main results are as follows:1.There were no significant differences in plant height,longest root length,fresh weight,dry weight and cadmium uptake between transgenic and non-transgenic rice seedlings under different concentrations of cadmium stress.2.The effect of low concentration(50mg/L)cadmium on the seed germination rate of rice was small.With the increase of cadmium concentration,the seed germination was inhibited more obviously.The seed germination rate was very low(less than 10%)under the high concentration(200mg/L)cadmium stress,and the seed ultimately died.The seed germination rate of transgenic rice was not significantly different from that of non-transge-nic rice under different Cd stress.3.The chlorophyll content of transgenic and non-transgenic rice seedlings decreased significantly with the increase of cadmium concentration.Under high concentration(200mg/L)cadmium stress,the chlorophyll content was very low,and even the transgenic rice leaves contained almost no chlorophyll.4.The contents of malondialdehyde(MDA)in aerial parts and root of rice seedlings were significantly increased with the increase of cadmium concentration,and there was no significant difference in MDA content between transgenic and non-transgenic rice seedings.