| Plant architecture breeding,aim to modifying the maize plant architecture,increasing plant destiny and improving the photosynthetic efficiency of population,will be the best way to achieve the goal of higher grain production.Leaf angel is the most important trait to ensure the plant architecture characteristic.Proper compact leaf angel can not only decrease the adverse effects of shade to gain the abilities of materials production and accumulation,but also enhance the ventilation and breathability of population to strengthen stress resistance,which plays an important role on improving the yield of maize.Therefore,gene cloning and molecular mechanism related to leaf angel has most important theoretical and practical significance for density tolerant and high yield breeding.This study focused on the main effective QTL q LA1 related to regulate leaf angel of maize.Firstly,we constructed the BC4F2?BC4F3 separation population by a cross from compact inbred lines Yu82 and loose inbred lines Shen137 for q LA-1 fine mapping and get the candidate gene in the region.Then we identified expression pattern by q RT-PCR and SAM technology and verify the function of the candidate gene by the maize genetic transformation.Finally,by using the yeast two hybrid system(Y2H)technology,we selected three genes interacting with Zm CLA1,and through a bimolecular fluorescence complementation(Bi FC)technology,the interaction between climate change was further to validate;Combined with cell morphology system,cell Morphological changes were analyzed by effects of candidate genes in the pulvinus of Shen137 and 137-NIL.This study has described systematically the function of Zm CLA1 on the molecular regulatory mechanism regulatory mechanisms during the formation process of maize leaf angle.The main results are as follows:1?By using Yu82 and Shen137 construction BC4F2?BC4F3 for fine mapping population contained 8752 individuals,and 7 pairs of polymorphic SSR markers were developed between LAI25 and maker bnlg1484.Athrough the method of molecular markers flanking,the q LA1-1 was finally defined between the markers LAII40 and LAII53,the interval distance was about 30.26 kb.Through the Maize GDB database and combined with the analysis of sequence differences,results showed that the GRMZM2G019260 gene as a candidate gene for q LA1-1,and named it Zm CLA1.2?Sequence analysis result of Zm CLA1 showed that the DNA sequence from the start codon to the stop codon was 9319 bp,and the sequence length of c DNA was 2097 bp,including 338 bp of the 5 ' untranslated regions(UTR)and 220 bp 3'-UTR and 1539 bp open reading frame(ORF),encoding a ERG8 kinase which was consisted of 512 amino acids containing GHMP kinase domain and Pmev-kin-ERG8 domain.Analysis of the sequence difference of parents and its near isogenic lines showed that the differences between 15 SNPs and 4 In Del sites in the promoter region before the start codon of 1250-1900 bp,and there is no difference between the coding regions.Difference analysis of the promoter sequences for the inbred lines which between the small leaf angle,Zheng58,Du321,Zheng32,B73 and large leaf angle,Yu 87-1,Mo17,Huang Zao4,Chang7-2,Shen5003.The results showed that the sequence of the inbred lines with smaller leaf angle were the same with yu82 and the large leaf angle were the same with shen137,indicating that these differences sites may be the main cause for the change of leaf angle.3?Real-time PCR was performed to determine the pattern of Zm CLA1 expression and the result showed that the accumulation of m RNA has a certain similarity between shen137 and shen137-NIL,both in the shoot apical meristem(SAM),leaf pulvinus,leaf sheath,leaf blade in plants.The accumulation has higher expression in these organs and the time were from 3 leaf stage,reached the peak at the leaf stage and a sharp decrease at the 11 leaf stage;and the accumulation of the m RNA in shen137 was significantly higher than of 137-NIL in different period and parts.In hybridization analysis showed that the accumulation of m RNA in shen137 was much higher than 137-NIL,the hybridization signals in 8 leaf stage was stronger than 7 leaf stage between shen137 and 137-NIL,these results tallies with the result of q RT-PCR.Combined with the dynamic change trend of leaf angle in shen137 and the results which leaf angle of shen137 is greater than shen137-NIL,implied that Zm CLA1 positive regulated the size of the leaf angle through the change of the accumulation of m RNA.4?Zm CLA1 overexpression and Zm CLA1 RNAi vectors were constructed and respectively introduced into the inbred Yu82 and shen137 through agrobacterium mediated shoot apex transformation technology.Between the sister line of T2 generation transgenic and non-transgenic line(control),the phenotypic analysis showed that leaf angle of the overexpressing(OX-CLA1)transgenic is increase 5.05 degree than the control;RNAi(RNAi-CLA1)strains of leaf angle mean value decreased 6.86 degree than the control,these results further provided that Zm CLA1 positively regulate the leaf angle.5?Using the yeast two hybrid system,we selected 38 positive clones interacting with Zm CLA1 from yeast library of Yu82,yeast by rotating the results showed that the gene interacted with protein kinase BAK1 involved in BR signal pathway,cysteine protease CYS regulating nucleic acid degradation and Auxin responsive protein IAA17.Bimolecular fluorescence complementation showed that BAK,CYS,IAA17 interacted with Zm CLA1 in vivo.These results indicate that Zm CLA1 through BAK,CYS,and IAA17 interaction may be involved in different pathways,regulation of maize leaf angle formation.Cytological observation showed that the support organization in Shen137-NIL located in the proximal part of leaf pulvinus developed more than that in Shen 137,which indicated that two materials of the proximal cells divided toward different directions: Shen137-NIL towards thick walled cells to erect leaves,and Shen 137 cells towards parenchyma in cell differentiation that has a weaker ability to support blade,which will lead to leaf angle increasing of Shen 137.In summary,Zm CLA1,as one cross node of two kinds of plant hormone signal pathway,mediates brassinosteroid(BR)and auxin(IAA)signal transduction in regulating leaf angle.Zm CLA1 participate in BR signal transduction pathway interaction with BAK1,which impact the proximal cells dividing toward different directions and further to change the leaf angel.At the same time interaction with the IAA17 effected on expression of Zm CLA4 and the auxin polar transport,which leaded to the uneven distribution of auxin and the changes of cell size,eventually leading to the changes of leaf angel.6?We used four RILs to analysis the leaf width QTL with the composite interval mapping method.The results is that we detected 46 QTLs of the leaf width above ear position in maize,the contribution to phenotypic variation for a single QTL varied from 4.33%-18.01%,there were 14 QTLs which the contribution rate was more than 10%.8 m QTLs for the leaf width of four RILs were detected with the method of meta-anlysis.The m QTL1-2,m QTL3-1,,m QTL7 and m QTL8 controlled two to four leaf width trait of different position and every m QTL included at least one QTL that the contribution rate was more than 10%.The results suggested there were key genes in these regions. |
PDF Full Text Request