| Rice (Oryza sativa L) is the most important food crops in China, approximately 65% of the national population depends on rice as staple food. Weeds can cause rice production declining dramatically or damaging the quality of rice. At present the most effective and economic method of controling weed in agricultural production is the use of chemical herbicides.Glyphosate is widely used in the fields weeding, for its high effective, low toxicity, low residue, non selective and weed killing spectrum. But because of glyphosate is a non selective herbicide, it will harm the crop directly used in the fields, and even cause them death.Therefore, cultivating the glyphosate-resistant rice is very important to increase the rice yield,reform the farming systems, and improve the labor productivity.In the process of research we found that when the transgenic rice from vegetative growth turn to reproductive growth,the expression of resistance gene in the stem tip is insufficient, so if the spraying time and application concentration is improper, the floral organ development will delay and even teratogenic lethality, ultimately affect the yield.So breeding the transgenic rice in which flower growth period have high expression of resistant gene OsEpspsM in the meristem is the key to the solution of glyphosate resistant transgenic rice practical.In this study,transgenic rice is homozygous line namely E6P14 carrying mutated gene EpspsM.We compared the field resistance and yield of transgenic lines when spraying herbicides in different periods.According to the expermental results we learned the tolerance concentration and drug safety period of the transgenic rice E6P14.We also isolated shoot tip specific promoter Lfy from Arabidopsis and flower development related gene promoter rfl from rice by homology cloning technology.And verified the specific regulation of the two promoters for gene expression.Using these two promoters we built five plant expression vectors,respectively is pCLfyGUS、pCAMBIA1300-Lfy-OsEpspsM、 pCAMBIA1300-Ubi-OsEpspsM and pCBIAM1300-Lfy-OsEpspsM-Ubi-OsEpspsM、 pCRflGVS,and obtained transgenic rice with these reconstructed vector by the genetic transformation method.Through a series of experimental analysis of the expression site specific promoter Lfy, Rfl in transgenic rice, and the ability of driving exogenous gene OsEpspsM. The results are as follows:1) Compared with the transgenic rice E6P14 with non spraying herbicide and wide 86 the average yield per plant was 27.26±0.76g which spraying the glyphosate (concentration of commodity 3 times the recommended concentration) after seeding 45 days, they have no significant difference at 0.05 level; Spraying glyphosate to the transgenic rice E6P14 which growth for 55 days, and the output is 22.95±1.78g at last.Compared it with E6P14 with non spraying (28.42±2.8g) and non transgenic rice 86 with non spraying(26.34±0.21g),its yield decreased significantly. The results show that after sowing 45 days with spraying the glyphosate, the rice yield was not affected; But in 45-55 days after sowing, the rice will be or has begun to enter the spike differentiation, transgenic rice on glyphosate sensitive, shoud do not spraying.2) We cloned the full length of the stem tip specific promoter Lfy from Arabidopsis thaliana, which is 2001bp long with a TATA-box upstream of the translatiom initiation codon-100～108;In addition to 4 CAAT-box which have funtion of enhancing and starting the transcription process. Furthermore there are many binging sites that relate to light response, such as GATA-motif、GAG-motif、 TCT-motif、GA-motif etc. We construct a plant expression vector pCLfyGUS and got 42 independent transgenic rice positive clones.By means of GUS histochemical staning and RT-PCR and Q-PCR detection on transgenic rice,we confirmed the stem specific promoter Lfy can drive foreign gene express specificly in panicles during the stage of spikelet differentiation of rice. We also built another plant expression vectors pCAMBIA1300-Lfy-OsEpspsM and pCAMBIA1300-Lfy-OsEpspsM-Ubi-OsEpspsM,and transformed them into rice,then we got 24 and 61 positive clones respectively. Detecting the panicle’ability to glyphosate by field resistance experiment and shikimic acid content of the transgenic rice before and after spraying glyphosate.The results demonstrate that the transgenic rice’ panicle which have double expression box Lfy-OsEpspsM-Ubi-OsEpspsM can resist three times higher than 13% glyphosate commercial propositional concentration.3) This study we cloned the full length of the promoter Rfl from rice,which is 2001bp long contains some TATA-box which is RNA polymerase binding site, In addition to there are some regulatory elments relate to floral organ development,for example AGAAA-box, CCWACC-box (W=A/T) and so on. Constructed the plant expression vector pCRf1GUS,and 72 strains transgenic clones by genetic transformation method. And it is proved that Rfl drive GUS gene in transgenic rice young panicle specific expression, by GUS histochemical staining and the mothed of Semi quantitative Q-PCR. |
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