Offspring Identification And Functional Analysis Of APX/8704/PPA Transgenic Upland Rice

Due to the severe environment,frequent disasters and the bottleneck of rice yield,the demand for stress-resistant rice varieties is increasing.As a variety of rice,upland rice has been paid more and more attention and utilized because of its small water demand and strong stress resistance.Therefore,optimizing the quality of upland rice has become a hot topic of agricultural scientific research in China.In this study,81 upland rice positive lines transformed with stress resistance genes APX,8704 and PPA are used as experimental materials,and their offsprings are identified by molecular analysis and functional analysis.Fifty-four candidate homozygous lines are identified by PCR molecular identification.Through field trait investigation and re-screening,21 candidate homozygous lines with excellent traits are selected,including 8dwarf lines and 13 high-quality panicle-type lines.Southern-Blot hybridization experiments are performed on these 21 candidate homozygous lines with these excellent traits,and 7 single-copy lines are obtained.Seven single-copy transgenic lines are amplified by Tail-PCR and their products are sequenced to obtain the insertion site results of six single-copy transgenic lines.The insertion point of the APX gene is 758392 bp of the base sequence of chromosome 5 in the high-quality panicle-type material APX trans-H1 strain F-4,and the insertion site is between Os05g0114000 gene and Os05g0114100 gene.The Os05g0114000 gene encodes similar to PRLI-like interaction factor,and the Os05g0114100 gene encodes similar to endonuclease/exonuclease/phosphatase family proteins.The insertion point of the PPA gene is 22012059 bp of the base sequence of chromosome 4 in the high-quality panicle-type material PPA trans-H2 strain H-40,F-36,F-38.And the insertion site is between Os04g0442000 gene and Os04g0442100 gene.The Os04g0442000 gene encodes similar to auxin-like response factor 2(ARF1 binding protein),and the Os04g0442100 gene encodes SMR protein/MutS2 C-terminal aldehyde main protein.The insertion point of the PPA gene is 24768677 bp of the base sequence of chromosome 7 in the short rod material PPA trans-H2 strain H-54,which is inserted into the coding gene Os07g0604700,which encoded a protein of the B12 D family.The upstream coding gene Os07g0604600 encoded similar to B12 D protein,and the downstream coding gene Os07g0604800 encoded similar to alpha-1,4-glucan-protein synthetase.The insertion point of PPA gene is29158910 bp of the base sequence of chromosome 4 in the dwarf material PPA transgenic H2 line H-55,and the insertion site is between Os04g0578400 gene and Os04g0578500 gene.The Os04g0578400 gene encodes similar to beta-ring hydroxylase,and the Os04g0578500 gene is a predictive gene.The determination of insertion sites proves that the exogenous genes have been successfully integrated into the upland rice genome.The determination of insertion sites and their upstream and downstream genes lays a foundation for later studies on the mechanism of action of the exogenous fragments integrated into the upland rice genome,as well as the development and utilization of transgenic lines.