The Study On Molecular Regulation Mechanism Of Increasing Lysine Content After Pyramiding O2, O16 And Wx Genes In Maize

To dissect molecular mechanism of increasing lysine content in maize through pyramiding high-lysine mutant genes?opaque2 and opaque16?and waxy mutant gene?waxy1?,using molecular marker-assisted selection technology,the o2 gene was introgressed into waxy maize using Taixi19/o2o2 as donor and QCL5013/wxwx as a receptor,we selected two o2o2 wxwx gene pyramiding lines,which were named QCL8004-1 and QCL8004-2.The o2 and o16 were introgressed into waxy maize using Taixi19/o2o2 and QCL3024/o16o16 as donor and QCL5019/wxwx as a receptor,we selected two o2o2o16o16 wxwx gene pyramiding lines,which were named QCL8006-1 and QCL8006-2.The o2,o16 and wx genes were introgressed into normal lines using three recessive gene mutant?QCL8006-1,o2o2o16o16wxwx?as donor and normal line?CML539?as a receptor,Seven types of NILs?o2o2o16o16wxwx,o2o2o16o16,o2o2 wxwx,o16o16wxwx,o2o2,o16o16,wxwx?were bred by multiple backcross and self-pollination.These mutants and their parent on kernels were subjected to RNA sequencing?RNA-Seq?,and revealed differentially expressed genes?DEGs?.Meanwhile,the label-free quantitation was used to analysis protein expression differences,as well as association analysis of transcriptome and protein.The key differentially expressed genes?DEGs?and proteins?DEPs?were integrated into metabolic pathways such as corn lysine,tryptophan and starch.These findings are of great importance for understanding the molecular mechanism underlying the lysine content increase due to pyramiding o2,o16 and wx allele.The main conclusions were as follows:1.Using molecular marker-assisted selection technology,QCL8004-1/o2 wx and QCL8004-2/o2 wx were bred by using Taixi19/o2o2 as donor and QCL5013/wx as a receptor,which the o2 gene was introgressed into waxy maize,and the background recovery rate based on SNP molecular markers was 93.16% and 93.28%,respectively.QCL8006-1/o2o16 wx and QCL8006-2/o2o16 wx were bred by using Taixi19/o2o2 and QCL3024/o16o16 as donor and QCL5019/wxwx as a receptor,which the o2 and o16 were introgressed into waxy maize,and the background recovery rate based on SNP molecular markers was 95.22% and 95.12%,respectively.Seven types of NILs?o216wx,o2o16,o2 wx,o16wx,o2,o16,wx?were bred by using three recessive gene mutant?QCL8006-1,o2o16wx?as donor and normal line?CML539?as a receptor,which the o2,o16 and wx genes were introgressed into normal lines,the theoretical of background recovery rate is 99.22%.It can be seen that these materials of different combinations of o2,o16 and wx genes are important materials for genetic research and breeding application of quality genes.2.In order to evaluate the nutritional quality of these materials,the amino acid contents were measured by automatic amino acid analyzers.The results showed that,compared with the recurrent parent QCL5013/wxwx,the lysine contents of QCL8004-1/o2o2 wxwx and QCL8004-2/o2o2 wxwx were 0.37% and 0.38%,and increased by 15.63% and 18.75%,respectively.Compared with the recurrent parent QCL5019/wxwx,the lysine contents of QCL8006-1/o2o2o16o16 wxwx and QCL8006-2/o2o2o16o16 wxwx were 0.49% and 0.54%,and increased by 68.97% and 86.21%,respectively.Compared with the recurrent parent CML539,the lysine contents of o2o16wx-NIL,o2o16-NIL,o2wx-NIL,o16wx-NIL,o2-NIL,wx-NIL and o16-NIL were increased by 111.22%,109.53%,91.22%,34.40%,79.93%,29.69% and 70.11%,respectively.3.In order to observe kernel characteristics and submicroscopic structure of these materials,Grain phenotypes and endosperm cross-sections of these mutant lines and their recurrent parent were observed under natural light,and the grain transparency was examined under projected light.The submicroscopic structure of these materials was observed by scanning electron microscopy?SEM?.The results showed that,grain seeds of the recurrent parent?QCL5013?and o2 wx mutant?QCL8004-1/o2 wx and QCL8004-2/o2wx?were smooth,opaque,and waxy.Meanwhile,there was a slight increase in the amount of farinaceous endosperm compared to the recurrent parent?QCL5013?.Under SEM,the starch granules of QCL8004₁ and QCL8004₂ had an irregular shape and arrangement,with a high density of matrix proteins that dispersed in the gap between starch granules,while those of QCL5013 were mostly elliptical and relatively smooth.Grain coats of o2o16 wx mutant lines?QCL8006-1/o2o16 wx and QCL8006-2/o2o16wx?were non-glossy and displayed different degrees of wrinkling;the grains were completely opaque,with farinaceous endosperm and no full kernels.Grain coats of recurrent parent QCL5019 were smooth and lustrous;the grains were opaque and full with waxy endosperm.Meanwhile,scanning electron microscopy revealed that the grain-endosperm starch granules of the o2o16 wx mutants had an irregular shape and arrangement and an uneven volume and size,with a high density of matrix proteins that dispersed in the gap between starch granules.The grain-endosperm starch granules of recurrent parent QCL5019 were mostly ellipsoid or spherical;the matrix-protein density was low,and the starch granules were closely encapsulated.The ear shape of NIls?o2o16wx-NIL,o2o16-NIL,o2wx-NIL,o16wx-NIL,o2-NIL,wx-NIL and o16-NIL?and recurrent parent CML539 were basically the same,and the grains were opaque and the degree of farinaceous endosperm was increased significantly.SEM revealed that the starch granules in endosperm of NILs were mostly irregular sharp,uneven in size and irregular in arrangement,with a high density of matrix proteins that dispersed in the gap between starch granules.In contrast,most of the endosperm starch granules of the recurrent parent?CML539?grains were ellipsoidal or spheroidal,with uniform size,low matrix protein density and tight packing of starch granules.4.Transcriptional analysis of waxy maize following the o2 gene introgression showed that,the O2 gene regulated multiple metabolic pathways related to BP and MP during waxy maize endosperm development.In the o2o2 wxwx kernels,Zm00001d029385 and Zm00001d012262,which encode the EF-1 and LHT1,respectively,were up-regulated,while Zm00001d011063,which encodes sulfur-rich proteins?mostly 10 kDa zein?,was down-regulated.These changes can improve the grain lysine content of waxy maize,although the functions of these genes in lysine biosynthesis or transport need to be further studied.Meanwhile,the O2 can directly or indirectly activate HSPs and EF2,and other DEGs associated with response to stimulus,the metabolic process,and other aspects of plant metabolism.These results are important for elucidating the underlying molecular mechanisms in the lysine content improvement of waxy corn following the o2 gene introgression.5.Transcriptional analysis of waxy maize following the o2 and o16 gene introgression showed that,272 genes were differentially expressed relative to recurrent parent by RNA-seq.GO and KEGG enrichment analyses revealed that these genes were mainly related to biomass metabolism.Among them,12 genes were enriched for amino acid metabolic pathways,including down-regulated lkr/sdh1 and Zm00001d020984.1,which inhibited the degradation of lysine.In addition,15 genes encoding ?-zein were down-regulated,which resulted in the reduction of ?-zein synthesis,while several non-zein proteins which accounts for most of the higher percentage of lysine are associated with varying degrees of increased accumulation.Furthermore,21 genes were related to carbohydrate metabolism;these included sh2,bt2,and ae1 genes upregulated,leaded to wrinkling kernel and farinaceous endosperm.These will help to uncover the transcriptional regulation mechanism in lysine content increased by o2 and o16 gene introgression into waxy maize.6.Transcriptional and protein analysis of normal maize following the o2,o16 and wx gene introgression showed that,under starch and sucrose metabolism,genes Shrunken2?Sh2?and Brittle2?Bt2?encoding ADP-glucose pyrophosphorylase?AGPase,EC2.7.7.27?in the conversion of ?-D-glucopyranose 1-phosphate to ADP-?-D-glucose in the starch synthesis pathway were up-regulated at 30 DAP,and the protein were up regulated,which was conducive to starch synthesis.Wx1 encoding granule binds the starch synthase?GBSS??GBE,EC 2.4.1.18?in the conversion of ADP-?-D-glucose to amylose in the amylose synthesis pathway were down-regulated at both 20 DAP and 30 DAP resulting in the inhibition of amylose synthesis.Zm00001d004438 encoding isoamylase?ISA,EC:3.2.1.68?in the conversion of ??1,6?-?-D-glucosyl-?1,4?-?-glucan highly branched to n?1,6?-?-D-glucosyl-?1,4?-?-glucan moderately branched in the amylopectin synthesis pathway was up-regulated at 30 DAP,and the protein was up-regulated resulting in the increased proportion of amylopectin.Under tryptophan metabolism,the related differentially expressed genes/proteins are mainly annotated into the tryptophan degradation pathway.Zm00001d011562 and Zm00001d039691 encoding the tryptophan pyruvate aminotransferase?TAA,EC: 2.6.1.99?in the conversion of tryptophan to indole-pyruvate in the tryptophan degradation pathway were down-regulated at 20 DAP and 30 DAP,and the protein were down regulated.Zm00001d025005,Zm00001d011764,Zm00001d023718 and Zm00001d044069 encoding indole-3-pyruvate monooxygenase?IPMO,EC: 1.14.13.168?in the conversion of indole-pyruvate to indoleacetic acid in the tryptophan degradation pathway,Zm00001d025005 and Zm00001d011764 were down-regulated 20 DAP and 30 DAP,Zm00001d023718 and Zm00001d044069 were down-regulated at 30 DAP,and the related protein Zm00001d023718.p001 is down-regulated at 30 DAP,inhibited the degradation of tryptophan.Under lysine biosynthesis metabolism,Zm00001d035282 encoding aspartic acid kinase?AK,EC: 2.7.2.4?in the conversion of L – aspartate to L-aspartyl-4-phosphate in the lysine synthesis pathway was up-regulated at 20 DAP and 30 DAP,promoted the synthesis of lysine.In lysine degradation pathway,Zm00001d052079 encoding amino acid lysine ketone reductase glutaric acid/yeast dehydrogenase?LKR/SDH??EC:1.5.1.8/1.5.1.9?in the conversion of lysine to?S?-2-aminoadipate-6-semialdehyde in the lysine degradation pathway,was down-regulated at 20 DAP and 30 DAP,and the protein was down-regulated at 30 DAP.In addition,Zm00001d020984 encoding L-pipecolate oxidase?L-PO,EC: 1.5.3.7?was down-regulated at 20 DAP and 30 DAP,and the protein was down-regulated at 20 DAP and 30 DAP,which inhibited the degradation of lysine and thereby increasing lysine content in seeds.