Creation And Characterization Of Transgenic High-starch,High Amylose Maize Germplasm

Maize is the most important crop cultivated worldwide with higher production than wheat and rice. Maize starch has the ideal structure and chemical property accounting for 80% of globle starch production. Starch serves as an indispensible component in human diet and an important raw material for both food and industries. The production, the components and structure of starch are key factors regulating the development of starch industry. In normal maize kernels, starch content is about 65%, and amylose accounts for 25-30% in total starch. Reinforcing the breeding of high-starch and high-amylose maize would achieve better economical and social benefit. However, in China there are few amylomaize germplasm and the supply of amylase mainly relys on import with high price. It is hard to break the linkage between some undesirable agronomic traits and the high amylose trait by conventional breeding methods. Therefore, it would be an efficient way to use transgenic strategy to obtain high-starch and high-amylose maize germplasm. The characterization of the progeny of SBEIIRNAi transgenic maize plantsAmylose synthesis is an multi-enzyme reaction and influenced by complicated gene-environment interactions. Starch branching enzymes (SBEs) play key roles in regulating the starch branching profile by catalyzing the cleavage of oligosaccharide chains through hydrolysis of the a-1,4 linkage and transferring it to another glucan chain to form branch points through a-1,6 linkages. Different SBE isoenzymes plays distinct roles in determining branching point number, chain length distribution and amylose to amylopectin ratio. The amylose content (AC) in endosperm is directly influenced by the activity of SBEIIb.In this work, we analyzed the genetic stability of the high amylose traits in these SBEIIRNAi transgenic plants and influence of SBEIIa and SBEIIb suppression on the real amylase content, chain length distribution, the starch granule morphology and the genetic stability of the high amylase trait. In the preliminary work, homozygous SBEIIRNAi transgenic maize lines have been obtained and identified by molecular detection, expression analysis and amylose content determination by Li et al.(2011). These 10 hairpin SBEIIRNAi vectors were designed targeting the ZmSBEII gene subfamily for co-suppressing the activity of SBEIIa and SBEIIb. These SBEIIRNAi vectors could be grouped into two sets in which the RNAi structures are promoted by the CaMV P35S or endosperm specific promoter P27kD. Within each set of SBEIIRNAi vectors, there were:two vectors containing the 893 bp (be) or 467 bp (bd) fragment within the ZmSBEIIb conserved domain (named P35bc, P35bd, P27bc and P27bd respectively); two vectors containing the fused fragment of 417 bp ZmSBEIIa specific domain and the 295 bp ZmSBEIIb specific domain (ac) or the fused fragment of 417 bp ZmSBEIIa specific domain and the 154 bp ZmSBEIIb specific domain (ad)(named P35ac, P35ad, P27ac and P27ad respectively); one vector contains the catalase intron replacing the original chalcone synthase intron (CHSA intron) in the vector containing the 893 bp ZmSBEIIb conserved domain (named P35in’and P27in’). The results from gel permeation chromatography (GPC) method indicated that among SBEIIRNAi transgenic lines, P27ac showed the highest amylose content, up to 55.89%, nearly two fold of WT plants. The amylose content of P27ad lines is 51.95%, obviously higher than that of lines P27bc and P27bd (46.24 and 44.78%, respectively). Lines P27bc, P27in’ and P27ad showed higher amylose content (46.24, 48.26 and 51.95%, respectively) than their corresponding P35bc, P35in’ and P35ad lines (41.86,45.31 and 46.83%, respectively). The amylose content in lines P35in’ and P27in’(45.31 and 48.26%, with catalase intron)were higher than that of P35bc and P27bc (41.86 and 46.24%, with CAHS intron). The amylose content was negatively correlated with the SBEII expression levels and SBE activity. These results consistent with the data of amylose content detected by idoin-binding method (Li et al.,2011). Meanwhile, the GPC results showed that the F1 component of SBEIIRNAi debranched starch contained more amylose moleculars with higher molecular weight. The ratio of F2 to F3 were greater than those from WT plants, suggesting that SBEIIRNAi maize starch contains more chains with longer chain length in amylopectin.In these SBEIIRNAi transgenic plants, along with the elevation of amylose content in endosperm, the starch granule exhibited obvious morghologic changes, such as irregular ellipsoidal shape and nonuniform size compared with the granules from nontransformed Chang7-2. The granules from transgenic lines containing the ZmSBEII conserved domain as the target sequence showed irregular protuberances, whereas starch granules from transgenic lines containing the ZmSBEII specific domain as the target sequence exhibited more concave morpha. This is suggesting that the alteration of starch composition have a direct impact on the starch granule morghology and the ratio of farinaceous endosperm to cutin endosperm in kernels. Compared with WT plants, the kernel size of SBEIIRNAi transgenic lines decreased and negatively related with the amylose content, but the kernels were not collapsed and retained the shape of their transformed receptor. Meanwhile, we analyzed the amylose content of the SBEIIRNAi T5 generation plants, which is similar with that of their T3 generation plants, indicating that the influence of SBEIIRNAi transgen on amylose content and the high-amylose trait could be stably inherited during each generations. Overexpression of mutated AGPase increased maize starch content and grain yieldAGPase is responsible for the production of ADPG and inorganic Pi by catalyzing the reaction between Glucose-1-Phosphate and ATP, providing the substrate ADPG of which the glucosyl group would be added to end of starch chains by starch synthase. AGPase is the key rate-limiting enzyme in regulating starch synthesis, and it is the target of genetic manipulation for improving starch accumulation. In maize, cytosolic AGPase is a hereotetramer composed of two large and two small subunits encoded by the Sh2 gene and the Bt2 gene respectively, and the activity is allosterically activated by 3-phosphoglyceride (3-PGA) and inhibited by inorganinc Pi. The large subunit encoded by the Sh2r6hs gene containing two mutant sites (a His-to-Tyr mutation at amino acide position 333 and an additional Tyr-Ser insertion), which could more stably interacted with the small subunit under high temperature and confer the AGPase reduced sensitivity to Pi suppression.In this work, we use splicing by overlap extension method obtaining the Sh2r6hs gene, which was introduced into maize elite inbred line Chang7-2 and Zheng58, solely and in tandem with Bt2 gene, under the control of the endosperm specific promoter P22kD or P27kD. PCR and real-time RT-PCR analysis showed that the transgen has been integrated into the genome of transgenic lines and it could stably expressed in the progeny plants. Homozygous transgenic lines were used to analyzed the influence of over-expression of Sh2r6hs and Bt2 on gene expression, AGPase activity, starch content, kernel phenotype and grain yield.We analyzed the gene expression and AGPase activity in endosperm from WT and T3 transgenic maize plants which were obtained from different grain-filling stages (10,15,20,25 DAP). It showed that the transcription levels of Sh2 and Bt2 genes and AGPase activity increased as the milking, and achieve the peak at 20 DAP, and then decreased. Compared with the WT plants, gene expression and AGPase activity were higher in transgenic plants and the Sh2r6hs and Bt2 double gene overexpression plants showed higher AGPase activity than Sh2r6hs single gene overexpression lines. Meanwhile, we analyzed the AGPase activity in endosperm at 20DAP under 45℃ incubation and the results showed that the activity of both WT and transgenic lines was lower than that under 30℃,but the activity of transgenic lines were higher than WT plants. Compared with the assay under 30℃, the AGPase activity of WT Chang7-2 plants retained only 64.99% while transgenic lines Sh2r6hsBt2-C-47 retained 77.59% and the WT Zheng58 plants retained 60.96% while transgenic line Sh2r6hsBt2-Z-80 retained 76.85%.The results showed that in Sh2r6hs sole over-expression transgenic lines starch content increased from 64.18% in WT plant to 70.4%. In double gene overexpression line Sh2r6hsBt2-C-47, the starch content was elevated to 77.05%, and in transgenic line Sh2r6hsBt2-Z-80, the starch content increased from 64.9%in WT Zheng58 to 77.36%. Meanwhile, compared with the corresponding nontransformed control, the 100-grain weight of transgenic lines was elevated by 14.6% (in Sh2r6hs-C lines),23.3% (in Sh2r6hsBt2-C lines) and 9.46% (in Sh2r6hsBt2-Z lines). The kernel size of transgenic maize plants also obviously increased compared with WT plants. Taken the data from pot and field experiments together, it showed that the growth, plant type, the row number and seed number of the ears from transgenic lines of the transgenic lines were similar with WT plant, but grain yield distinctly increased, which was mainly resulted from the increase of 100-grain weight.In this study, single Sh2r6hs gene over-expression or co-expression with the Bt2 gene could increase the AGPase activity, starch content and grain yield, and the double genes over-expression could result in a higher elevation. It was deduced that in the single over-expression lines, the elevation of AGPase activity was achieved through promoting the subunit-hereotetramer balance shifting to the hereotetramer forming direction and the mutant large subunit could more efficiently recruit the small subunit to form stable hereotetramer; in the double genes over-expression lines, there were more stable hereotetramer which could defense the heat stress and reduce the impact of Pi inhibitor. The increase of AGPase activity could provide more ADPG for starch synthase, and facilitate the soluble sugar to starch conversion, which could draw more carbohydrates to from the "source" tissue to the "sink" tissue, resulting in a grain yield increase. In this work, we successfully applied the strategy of over-expressing the mutant AGPase coding gene in maize endosperm and obtained transgenic lines with increased starch content and grain yield, providing a useful maize germplasm for further high-starch maize breeding practice. Over-expression of GBSSI increased amylose content in maize kernelGBSSI is the unique starch synthase that could synthesize amylose in maize endosperm, which could add the glucose from ADPG to the non-reducing end of amylose or brancing chains of amylopectin. The loss of GBSSI function will resulted in a sever decrease in amylose content, even in some mutants, the starch consisted 100% amylopectin. Therefore, over-expression of the ZmWx gene encoding the GBSSI in maize endosperm would increase the GBSSI activity and might improve amylose synthesis.In our work, ZmWx gene was cloned from the cDNA of maize endosperm and was introduced into elite maize inbred line Chang7-2 and Zheng58 under the control of P27kD. PCR and real-time RT-PCR analysis showed that the transgen has been integrated into the genome of transgenic lines and it could stably expressed in the progeny plants. Homozygous transgenic lines were used to analyzed the influence of over-expression of ZmWx gene on gene transcription, GBSSI activity, amylose content, kernel phenotype and grain yield.We analyzed the gene expression and GBSSI activity in endosperm from WT and T3 transgenic maize plants which were obtained from different grain-filling stages (10,15,20,25,30,35 DAP). It showed that the transcription levels of ZmWx gene increased as the milking, and achieve the peak at 30 DAP, and then decreased, while GBSSI activity achieve the peak at 35 DAP. Compared with the WT plants, gene expression and GBSSI activity were higher in transgenic plants. We measured the amylase and starch content of the homozygous transgenic lines and WT plant. The results showed that all the transgenic lines have higher AC than that of WT plants. The AC was increased from 25.3% in WT Chang7-2 plants to 41.1% in in transgenic lines Wx-C-5 and the AC was elevated from 26.4% in WT Zheng58 plants to 41.9% in transgenic lines Wx-Z-20. Meanwhile, the starch content of transgenic lines were increased to 71% compared with the 64.18% in WT Chang7-2 or 66.21% in WT Zheng58 plants.100-grain weight showed a distinguishing increase about 9.06% in Wx-C lines and 14.97% in Wx-Z compared with wild type plants. The data from both pot and field trial showed that Zm Wx transgenic lines showed normal growth and the row number per ear is similar with ear from WT plant, however, the ear length, seed per row,100-grain weight and grain yield showed distinct elevation compared with their corresponding WT plant.In our work, the endosperm-specific over-expression of ZmWx gene resulted in a elevation of GBSSI activity, which means there were more GBSSI protein playing effective roles in the endosperm cells. Unlike other starch synthase, GBSSI was fixed in the starch granule as the granule growing. Therefore, it was an efficient way to improve the amylose synthesis by increasing the abundance of GBSSI protein in the semi-crystalline structure. Meanwhile, accompanying AC improvement, the starch content and seed weight were not decreased but slightly increased, it was speculated that GBSSI not only could synthesize amylose, but also could acting on the side chains in amylopectin in charge of the extra-long chains synthesis, thus the GBSSI over-expression could also improve total starch synthesis and promote carbohydrate acceleration in kernels leading to a grain yield increase.Characterization of mutant AGPase and GBSSI co-overexpression transgenic pyramiding maizeConsidering that except for GBSSI protein levels, the ability of GBSSI to synthesize amylase was also contorl by the concentration of substrate ADPG. In this work, we conducted the transgenic pyramiding between ZmWx and Sh2r6hs-Bt2 over-expression transgenic lines to investigate whether the amylose synthesis could be improved to higher levels. The ZmWx, Sh2, Bt2 gene expression, AGPase and GBSSI activity in endosperm from transgenic pyramiding plants were higher than that from WT plants at different grain-filling stages (10,15,20,25,30,35 DAP). In the transgenic pyramiding plants taking Sh2r6hs-Bt2 over-expression plants as female parent, the expression of Sh2 gene and Bt2 gene were higher than that from the reciprocal cross plants. Similarly, the ZmWx transcription level and GBSSI activity were higher in the pyramiding plants from GBSSI over-expression plants as the female parent than that from the reciprocal cross plants. For the time limited, the exogenous Sh2r6hs、Bt2 and ZmWx were not homozygous in the transgenic pyramiding plants. In the genome of endosperm from parental plants, there were three copies of exogenous transgen, but in that of transgenic pyramiding plants, there were only one or two copies of each exogenous transgen. Therefore, influenced by this gene dosage effect, we could not perorate whether amylose synthesis could be improved to higher levels by co-overexpression of the GBSSI and mutant AGPase.In conclusion, we analyzed the changes of starch structure and starch granule in high amylose SBEIIRNAi transgenic maize plants, and further affirmed the suppression of SBEII genes by different RNAi structures; we obtained mutant AGPase over-expression transgenic lines with higher starch content and seed weight; for the first time we reported that ZmWx over-expression could improve the amylose synthesis, obtaining transgenic lines having higher amylose and starch content, and we got transgenic pyramiding plants co-overexpression mutant AGPase and GBSSI, providing new germplasm for further high-starch high-amylose maize breeding.