| Maize is one of the most important food and feed crops, which has a special attraction as an industrial raw material in food and chemical industries, and plays an important role in human life. Production of starch around the world is 3,600 tons every year, of which more than 80% is corn starch, therefore strengthening the high-starch corn breeding has significant social and economic benefits. Amylose is a kind of important industrial raw material, widely used, relate to various fields, such as food, medical, textile, paper, packaging, petroleum, environmental protection, optical fiber, a high degree of printed circuit boards and electronic chips and other industries. But amylose content of normal corn is between 22-28%, and it is costly to extract amylose from normal corn. At present, the amylose in China relies mainly on imports from America, with high price. The synthesis of amylose is affected by complex gene environmental interactions, and increased amylose content is usually accommpanied with the reduction of the yield and other agronomic traits. Varieties of high amylose corn have been bred by using conventional breeding techniques, of which the main problems are the reduced total content of starch, higher moisture content and the final cut, while genetic engineering technology is a way worth exploring for high-amylose maize cultivation.Over-expression of AGPase genes increased starch content and corn grain yieldStarch is mainly synthesized in amyloid and the chloroplast, the whole process is controlled by a series of enzymes. ADP-glucose pyrophosphorylase is the key factor of starch synthesis pathway, playing a major role in regulating the starch synthsis, which converts glucose 1-phosphate into the substrate ADPG for starch synthesis. Over-expression of AGPase can improve the AGPase activity and increase the starch content. AGPase is a tetrameric enzyme that consists of two large subunits and two small subunits in maize, playing an important role in maize seed development and starch synthesis.Maize genes encoding AGP large subunit Sh2 and small subunit Bt2 were cloned by RT-PCR from the maize endosperm cDNA, and expression vectors were constructed for over expressing the two genes (Sh2, Bt2), solely and in tandem, driven by maize endosperm-specific promoters, with herbicide resistance gene as a selectable marker. These three expression vectors were transformed into maize elite inbred lines Ye478 and Chang7-2, by Agrobacterium-mediated genetic transformation of maize bud tip method. PCR and Southern testing confirmed that the foreign genes had been integrated into transgenic maize genome and stably expressed in the offspring. Lines from different independent transformants with single copy of the foreign gene inserted were selected for further study of transgene expression, starch synthesis, grain yield and other agronomic traits.Analysis of gene expression and enzyme activity were performed in WT and transgenic maize of T3 generation, using kernels obtained at different filling stages (10,15,20,30 DAP), and starch content analysis and biomass measurement were detected with mature seeds. It showed that the expression of AGPase gene and enzyme activity increased as time, and reached a peak at the 20DAP, then decreased slowly. Compared with the non-transgenic plants, gene expression and enzyme activity of AGPase have increased in all transgenic plants. Elevation of AGPase activity in plants over expressing Sh2 or Bt2 were less than in plants with double genes over expressed in series, while changes between plants with single gene over expression were little. Changes of starch content analysis in mature seeds were consistent with the trend of gene expression and enzyme activity. The starch content increased to 71-74% in single gene transgenic lines, compared with the 66% (Chang 7-2) or 65% (Ye 478), respectively; in the double gene series lines, starch content increased to 76-78%, about 16.7% over the wild type.100 grain weight also had significant increase in transgenic plant, there were about 20% (Bt2),18% (Sh2) and 30% (Bt2Sh2) increase compared with non transgenic plant Chang 7-2, and 22% (Bt2),20% (Sh2) and 33% (Bt2Sh2) increase compared with non transgenic plant Ye478. Rsearch and observation of pot and field experiments showed that the transgenic plants had normal growth, the ear length, row numbers per ear and kernel number had no significant difference compared with wide type plants, but ear weight, kernel weight and starch content were significantly higher than non-transgenic plants, and kernal size also had remarkbally increased, especially in double genes transgenic plants.In this study, the use of the endosperm-specific promoter, zein gene promoter, greatly enhanced AGPase gene expression in maize endosperm. Single-gene over-expression of Bt2 or Sh2 can also increase AGPase activity, while over-expression of the Bt2 and Sh2 genes together enhanced the AGPase activity to a significantly higher level. The increase of AGPase activity could be the result of the increase of tetramers in the endosperm cytoplasm and the higher stability of the tetramers. The increase in AGP activity in the filling endosperm could enhance the sink activity in developing seeds and facilitate the conversion and translocation of carbohydrates. This could speed up photosynthesis by drawing more sugars into the developing seeds. Thus, the seed weight and yield would be improved. It is possible that we could produce maize varieties with better photo synthetic efficiency, higher grain yields, and higher starch content by enhancing the AGPase activity in the endosperm cytoplasm. Our work successfully provided a practicable way to breed good maize inbred lines.RNAi mechanism created new materials of high-amylose maize linesThe synthesis of amylose is controlled by several genes, and complex gene interactions are also involved in regulating endosperm amylose content. Increase in amylose content of endosperm is often accompanied with decrease of yield and other agronomic traits. Starch branching enzyme is an important regulatory enzyme of starch biosynthesis in plants, which can hydrolyses an a (1,4) linkage between polymers, and link the short-chain to the receptor chain by an a (1,6) glycosidic linkage to form a branching structure. Thereby inhibiting the expression of starch branching enzyme gene can affect intracellular enzyme content or activity, reduce amylopectin synthesis, and increase amylose content. There are three isoforms of starch branching enzymes in maize, SBEI,SBEIIa and SBEIIb, while SBEIIb plays the most important roles on amylase content of maize endosperm.The specific area and conservative fragment of corn starch branching enzyme SBEIIa and IIb were cloned by RT-PCR method from the maize endosperm cDNA. Plant expression vectors of RNAi for the SBEIIa, SBEIIb were constructed, promoted by maize endosperm-specific promoter p27Kd or the constitutive promoter P35S. In order to obtain optimum interference efficiency, and greatly increase the amylase content to meet the needs of the plastics industry, different fragments of conservative areas and specific areas of the SBEIIa, SBEIIb gene were selected for the target. Based on the SBEIIa and SBEIIb conserved sequence, RNAi structures were constructed with the hpRNA arm length of 893bp and 467bp; based on the SBEIIa specific sequences,417bp or 154bp, were respectively connected to the SBEIIb specific sequence of 295bp to construct different hpRNA structures; different introns and promoters were used to construct different RNAi specific structures. By conventional molecular biology techniques, these RNAi structures were restructured to the plant expression vectors, and obtained 10 plant expression vectors with the different characteristics. These ten different RNAi structures were transformed into maize elite inbred lines Chang7-2, by Agrobacterium-mediated genetic transformation of maize bud tip method. Transgenic lines of 7 RNAi structures were obtained. Herbicide selection, PCR and gene expression analysis indicated that the RNAi structures had been integrated into transgenic maize genome and stably expressed in the offspring.Lines from different independent transformants with single copy of the foreign gene inserted were selected for further study. The agronomic traits, gene expression intensity, SBE activity, total starch content, amylose content of endosperm were analyzed. These results showed that structures of RNAi effectively inhibited the SBEII gene expression, significantly reduced the activity of the endosperm SBE, and the amylose content improved significantly, compared with wild-type control. Plant growth and development morphology had no significant change, but the total starch content and grain yield had a certain reduction.SBEII gene expression and SBE enzyme activity analysis at filling stage of 20DAP showed that in RNAi transgenic plants, SBEII gene expression and SBE enzyme activity decreased to varying degrees, and interference by 27Kd promoter in transgenic plants were more efficient than the 35S series; between RNAi structures with the different lengths of SBEIIb conserved sequences, the interference efficiency is not very different; interfere with both specific area of SBEIIb and specific area of SBEIIa, interference efficiency was most obvious in all carriers; RNAi structure with a short intron, inhibition efficiency is relatively high.Amylose and total starch content were measured in mature seeds endosperm of plants with RNAi structures and control plants. It showed that with the RNAi structures based on SBEIIb conserved regions of different arm length, amylose content of seed endosperm in transgenic plants had no significant difference to each other; while with the RNAi structures based on the connection of SBEIIa and SBEIIb specific sequence, RNAi efficiency seemed better with arm length of 712bp than 349bp; In plants with RNAi structures based on specific sequence of SBEIIa and SBEIIb connection, increase of amylose was greater than those based on SBEIIb conserved sequences. Replacing pFGC5941 vector intron with catalase intron vector allowed more amylose synthesis. In plants with RNAi structures promoted by P27Kd, the amylose content was higher than those promoted by P35S, and the growth and morphology of former transgenic plants were more similar to WT control. In lines which were interfered by both specific area of SBEIIb and SBEIIa, amylose content increased to 55% (pot) or 54% (field), while the total starch content reduced to less than 60%,10% lower than 65% of non-transgenic control, and the 100 grain weight is only 17g, about 84% of the control of non-transgenic (20.2g/100 tablets). Inhibiting of the activity of SBE, the overall level of starch synthesis may have been somewhat suppressed, which resulted in lower starch content and reduction of 100 grain weight.Through research and observation of field experiments, plant height, ear height, ear length, single ear weight, and other agronomic traits showed that the transgenic plants had no significant difference with wide type plants; only in the plants with 35S promoter-driven RNAi structures, plant height and leaf area were lower than in the wild type.This work has obtained a new high amylose maize material, and accumulated important data for RNAi structure design and high amylose corn creation through transgenic technology.Obtain the double high maize by hybrid of high-starch transgenic maize and high-amylose transgenic maizeUsing RNAi approach, we obtained maize lines with high amylos 50%-55%, while the total starch content was lower than that of wild-type control. In order to solve the problem of total starch content decrease and the decline in output, we introduced the expression vector structure accelerating the rate-limiting step in starch synthesis to transgenic lines with high amylose, to increase the AGPase activity in receptors, and promote more carbohydrates in plant and endosperm to flow to starch synthesis, which to some extent, increase the starch content and yield. In this work, the materials used for hybrid selection switch to the highest starch content transgenic plants with two-gene of AGPase subunits tandemly expressing, and high amylose lines ac, which interfere both specific areas of SBEIIa and SBEIIb, genotype of plant material are Chang 7-2.3 homozygous transgenic lines with high starch were selected as male parent, and 3 homozygous transgenic lines with high amylase were selected as female parents. The transgenic lines were hybridized in pair, to obtain the F1 generation. Southern hybridization showed that AGPase gene over-expression structure and RNAi structure were both exited in the polymerization plant genome.Seeds of Fl generation of transgenic polymerization plants with AGPase gene over-expression and RNAi structure, seeds of sister crossing of non-transgenic plants control, seeds of sister crossing of high starch corn and sister crossing of high amylose corn control were planted in pot of the same size, growing in suitable conditions and self-pollination. Gene expression and enzyme activity were determined at developmental stages (20DAP). Total starch content and amylose content of the dry mature seeds were measured and agronomic traits, inducing plant height, biological characteristics such as 100 grain weight were observed at the same time.In the endosperm of transgenic polymerization lines, the expression level of Bt2, Sh2 and AGPase activities were slightly lower than the male parent plant, but still significantly higher than wild-type control; expression level of SBEII was slightly higher than the female parent, but not significantly, still lower than the wild-type control, and SBE activity showed the same trend. It shows that in the polymerization transgenic plants, over-expression of AGPase and interference of SBE both have a certain degree of performance. Although amylose content in transgenic hybrid polymer declined a little compared with the female parent, but still higher than 50%; the total starch content increased to 71% compared with non-transgenic control of 66%, though lower than father parent of 78%. Hybrid polymer plants had normal growth in the development process, plant height, ear height, ear length and grain phenotype did not change significantly compared with non-transgenic plants.100 grain weight increased by over 18% than the high amylose plants with RNAi structure vector, similar to or slightly higher than control plants Chang 7-2. The results showed that the hybrid polymer transgenic plants not only exhibited high amylose content, but also had the higher total starch content, providing an effective method to solve the problem of low starch content in the female parent plants.In summary, it is the first report to obtain high-starch and big grain size maize by over expression of AGPase genes Bt2 and Sh2 in tandem; High amylose maize plants with amylose content over 50% were obtained through RNAi of SBEII, and interference efficiency of different RNAi structures were compared, which provided a reference for RNAi vector construction design and improvement of RNAi efficiency in plants; A new material corn of high starch, high amylose was obtained by hybridization of transgenic plants with over-expressing of AGPase genes and RNAi of SBEII genes. The work was a useful attempt to foster high-starch corn, high amylose corn and high-starch high-amylose corn of great economic value, and provided effective ways and methods for the cultivation of high-starch high-amylose products.
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