农杆菌介导的IPT和PEPC基因共转化台粳9号水稻的研究
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摘要
大多数陆地植物利用C_3光合途径中的核酮糖1，5-二磷酸羧化酶／加氧酶(Rubisco)吸收大气中的CO_2。C_4植物除了C_3途径外，还利用C_4光合循环在Rubisco位点提高CO_2浓度，其中一个关键酶是磷酸烯醇式丙酮酸羧化酶(PEPC)，它与CO_2的亲和性比Rubisco高。与C_3植物相比，C_4植物的光合效率提高近2倍。因此长期以来人们期望将C_4特征转移到C_3植物上提高C_3植物的光合性状。
水稻生育后期叶片过早衰老，导致光合效率降低。有报道指出，延缓一天衰老，生物产量可提高2％。细胞分裂素是一种重要的延缓植物叶片衰老进程的植物激素，异戊烯基转移酶(IPT)基因是其生物合成的关键酶基因。将叶片衰老相关的启动子(SAG1和SAG2)与IPT基因融合构成嵌合基因pSAG12-IPT后再导入水稻中，可以特异性地提高转基因水稻体内的细胞分裂素水平，从而有效地延缓水稻叶片的衰老。
本研究初步建立了台粳9号的再生体系，采用根癌农杆菌介导的遗传转化系统，将已经构建好的嵌合基因pSAG12-IPT和无标记的pCAMBIA1300-Ubi-PEPC-NOS，通过双菌共转化的方法导入优良水稻品种台粳9号幼胚诱导的胚性愈伤组织中，经潮霉素筛选，抗性愈伤分化成苗，共获得36个抗性愈伤克隆，41株转基因植株，对这些植株进行PCR检测和GUS染色分析，确定其中16株为阳性植株，10株为含IPT基因阳性植株，9株为含PEPC基因阳性植株，3株既含有IPT基因又含有PEPC基因，共转化率达18.75％。
在水稻遗传转化的同时，将玉米的完整PEPC基因(包括所有外显子、内含子、启动子和终止子，共8.5 kb)构
建到植物双元表达载体上pCAMBIA 1 300。拟在获得无标
记PEPC基因的水稻植株基础上进行第二次遗传转化。
通过对同一批16株转基因水稻植株的光合速率进行
测定，结果表明，在所有转IPT基因的水稻植株中，有8
株的光合速率比对照高6.26%一73.22%，有2株比对照低
9.17%和20.67%。在所有转PEPC基因的水稻植株中，有7
株的光合速率比对照高48.76%~63.46%，有2株比对照低
16.89%和31.88%。在既含有I户T基因又含有PEPC基因的3
株转基因水稻的光合速率均比对照高，最高的一株比对
照高88.65%。
The majority of terrestrial plants assimilate atmospheric CO2 through the C3 photosynthetic pathway, and the enzyme of primary CO2 fixation in this pathway is ribulose 1,5-bisphosphate carboxylase/oxygenase
(Rubisco). In addition to the C3 pathway, C4 plants use the C4 photosynthetic cycle to elevate the CO2 concentration at the site of Rubisco. One of the key enzymes in C4 pathway is phosphoenolpyruvate carboxylase (PEPC) that has higher affinity for CO2 than Rubisco. The C4 plants have up to twice photosynthetic rate than C3 plants. Consequently, it has long been expected that the transfer of C4 traits to C3 plants could improve the photosynthetic performance of C3 species.
The photosynthetic rate decreases due to leaf senescence during the late growth period of rice. It is reported that the biomass can be elevated by 2% if the senescence can be delayed for one day. Cytokinin is an important phytohormone involved in delaying senescence of plant leaf, and isopentenyl transferase (IPT) gene encodes the key enzyme that catalyzes cytokinin biosynthesis. Transformation of chimeric gene which consists of leaf-specific promoter (SAG 1 and SAG 2) of the senescence expression and IPT gene can specifically increase cytokinin of transgenic rice and thus delay the leaf senescence effectively.
The regenerating system of rice cultivar Tai 9 is established in this experiment. Using an Agrobacterium-mediated transformation system, we have
introduced both the chimeric gene pSAG12-IPT and marker-free pCAMBIA1300-Ubi-PEPC-NOS into the embryogenic calli of elite japanica rice variety Taijing 9 through co-transformation method. The calli were selected on the NB media plus with hygromycin. And then 36 clones of calli resistant to hygromycin were obtained and differenciated into the plantlets. A total of 41 primary transgenic rice plants were obtained from antibiotic-resistant cells. Sixteen transgenic rice plants were determined to be positive through histochemical GUS assay and PCR assay, and 10 rice plants contained IPT gene and 6 rice plants contained PEPC gene, and 3 plants contained both IPT and PEPC gene among them. The co-transformation rate was 18.75%.
The maize intact PEPC gene (8.5 kb) including all exons, introns, promoter and terminator was constructed on plant binary expression vector pCAMBIA1300 during rice genetic transformation. The second genetic transformation will be conducted on the basis of obtaining marker-free transgenic rice plant containing PEPC.
Photosynthetic rates of 16 transgenic rice plants were measured under ambient conditions. The results showed that 8 of them were higher 6.26%~73.22% and 2 of them were lower 9.17% and 20.67% tha.n that of the control among all transgenic rice containing IPT gene, and that 7 of them were higher 48.76%~63.46% and 2 of them were lower 16.89% and 31.88% than that of the control among all transgenic rice containing PEPC gene in terms of photosynthetic rate. The transgenic rice plants containing both IPT and PEPC gene had a higher photosynthetic rate
than that of control and the highest one was improved by 88.65%.
水稻生育后期叶片过早衰老，导致光合效率降低。有报道指出，延缓一天衰老，生物产量可提高2％。细胞分裂素是一种重要的延缓植物叶片衰老进程的植物激素，异戊烯基转移酶(IPT)基因是其生物合成的关键酶基因。将叶片衰老相关的启动子(SAG1和SAG2)与IPT基因融合构成嵌合基因pSAG12-IPT后再导入水稻中，可以特异性地提高转基因水稻体内的细胞分裂素水平，从而有效地延缓水稻叶片的衰老。
本研究初步建立了台粳9号的再生体系，采用根癌农杆菌介导的遗传转化系统，将已经构建好的嵌合基因pSAG12-IPT和无标记的pCAMBIA1300-Ubi-PEPC-NOS，通过双菌共转化的方法导入优良水稻品种台粳9号幼胚诱导的胚性愈伤组织中，经潮霉素筛选，抗性愈伤分化成苗，共获得36个抗性愈伤克隆，41株转基因植株，对这些植株进行PCR检测和GUS染色分析，确定其中16株为阳性植株，10株为含IPT基因阳性植株，9株为含PEPC基因阳性植株，3株既含有IPT基因又含有PEPC基因，共转化率达18.75％。
在水稻遗传转化的同时，将玉米的完整PEPC基因(包括所有外显子、内含子、启动子和终止子，共8.5 kb)构
建到植物双元表达载体上pCAMBIA 1 300。拟在获得无标
记PEPC基因的水稻植株基础上进行第二次遗传转化。
通过对同一批16株转基因水稻植株的光合速率进行
测定，结果表明，在所有转IPT基因的水稻植株中，有8
株的光合速率比对照高6.26%一73.22%，有2株比对照低
9.17%和20.67%。在所有转PEPC基因的水稻植株中，有7
株的光合速率比对照高48.76%~63.46%，有2株比对照低
16.89%和31.88%。在既含有I户T基因又含有PEPC基因的3
株转基因水稻的光合速率均比对照高，最高的一株比对
照高88.65%。
The majority of terrestrial plants assimilate atmospheric CO2 through the C3 photosynthetic pathway, and the enzyme of primary CO2 fixation in this pathway is ribulose 1,5-bisphosphate carboxylase/oxygenase
(Rubisco). In addition to the C3 pathway, C4 plants use the C4 photosynthetic cycle to elevate the CO2 concentration at the site of Rubisco. One of the key enzymes in C4 pathway is phosphoenolpyruvate carboxylase (PEPC) that has higher affinity for CO2 than Rubisco. The C4 plants have up to twice photosynthetic rate than C3 plants. Consequently, it has long been expected that the transfer of C4 traits to C3 plants could improve the photosynthetic performance of C3 species.
The photosynthetic rate decreases due to leaf senescence during the late growth period of rice. It is reported that the biomass can be elevated by 2% if the senescence can be delayed for one day. Cytokinin is an important phytohormone involved in delaying senescence of plant leaf, and isopentenyl transferase (IPT) gene encodes the key enzyme that catalyzes cytokinin biosynthesis. Transformation of chimeric gene which consists of leaf-specific promoter (SAG 1 and SAG 2) of the senescence expression and IPT gene can specifically increase cytokinin of transgenic rice and thus delay the leaf senescence effectively.
The regenerating system of rice cultivar Tai 9 is established in this experiment. Using an Agrobacterium-mediated transformation system, we have
introduced both the chimeric gene pSAG12-IPT and marker-free pCAMBIA1300-Ubi-PEPC-NOS into the embryogenic calli of elite japanica rice variety Taijing 9 through co-transformation method. The calli were selected on the NB media plus with hygromycin. And then 36 clones of calli resistant to hygromycin were obtained and differenciated into the plantlets. A total of 41 primary transgenic rice plants were obtained from antibiotic-resistant cells. Sixteen transgenic rice plants were determined to be positive through histochemical GUS assay and PCR assay, and 10 rice plants contained IPT gene and 6 rice plants contained PEPC gene, and 3 plants contained both IPT and PEPC gene among them. The co-transformation rate was 18.75%.
The maize intact PEPC gene (8.5 kb) including all exons, introns, promoter and terminator was constructed on plant binary expression vector pCAMBIA1300 during rice genetic transformation. The second genetic transformation will be conducted on the basis of obtaining marker-free transgenic rice plant containing PEPC.
Photosynthetic rates of 16 transgenic rice plants were measured under ambient conditions. The results showed that 8 of them were higher 6.26%~73.22% and 2 of them were lower 9.17% and 20.67% tha.n that of the control among all transgenic rice containing IPT gene, and that 7 of them were higher 48.76%~63.46% and 2 of them were lower 16.89% and 31.88% than that of the control among all transgenic rice containing PEPC gene in terms of photosynthetic rate. The transgenic rice plants containing both IPT and PEPC gene had a higher photosynthetic rate
than that of control and the highest one was improved by 88.65%.
引文
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