| Chlorophyll is a key pigment for plants to capture light energy and perform photosynthetic functions.Chlorophyll degradation is the most obvious sign of plant leaf senescence.The decline of assimilation function of plant leaves caused by senescence will greatly limit the development of crop yield potential.Chlorophyllase is one of the key enzymes in the process of chlorophyll degradation,it can hydrolyze chlorophyll a into phytochlorophyll a.Previous studies have found differences in the role of chlorophyllase genes in different crops or different varieties of the same crop or different family members in the aging process.The chlorophyllase gene of wheat includes chlorophyllase 1 and chlorophyllase 2,but their’s role in aging is unknown.Therefore,cloning wheat chlorophyllase 1 gene and analyzing its expression characteristics during leaf senescence can lay the foundation for further research on its function and role.In this study,we used the wheat variety Zhong Mai 175 as the material to isolate and identify the chlorophyllase 1 gene(TaCLH1)from the three genomes of A,B,and D,and performed sequence polymorphism analysis and molecular marker development.At the same time,we analyzed its expression characteristics during different hormone treatments and natural senescence;determined the chlorophyll a and chlorophyll b content and SPAD value of wheat leaves during natural senescence,and inferred its role in chlorophyll degradation through comparative analysis.The main findings are as follows:1.We cloned the three homologous gene coding sequences of wheat TaCLH1,which were 1118 bp,1161 bp,and 1126 bp in length,and were located on the short arm of the sixth chromosome.The homology between them was 93.26%.The analysis of gene structure showed that all the three homologous genes contained two exons and one intron,encoding 315,317 and 319 amino acids respectively.Conserved domain analysis revealed that they all encode highly conserved α / β hydrolase superfamily domain proteins.2.We used 32 materials with high polymorphism to analyze the sequence polymorphism of three homologous genes of TaCLH1.We found that the sequences of TaCLH1-6A and TaCLH1-6D were conservative,and there was no polymorphism site.However,the sequence polymorphism of TaCLH1-6B was high,21 SNPs and 5 In Del were found,forming three haplotypes,TaCLH1-6B-Hap1,TaCLH1-6B-Hap2,TaCLH1-6B-Hap3.Two caps molecular markers,TaCLHI-6B-Sac Ⅱ-CAPS and TaCLH1-6B-Nsi Ⅰ-CAPS,were developed based on polymorphic loci to distinguish three haplotypes.3.Analysis of the expression characteristics of TaCLH1 s gene in different organs found that the expression level in leaves was the highest,and that in other organs was lower;The treatment of wheat leaves with ABA and Me JA found that the expression of TaCLH1 s gene reached the highest at 12 h,then decreased,and finally returned to the level before treatment;It was found that the expression of TaCLH1 s gene in wheat leaves under SA treatment did not follow certain rules;The 6-BA treatment of wheat leaves found that the expression of TaCLH1 s reached the lowest at 6 h,and reached the lowest at 12 h during dark treatment,then increased,reached the maximum at 24 h,and finally decreased to the pre-treatment level;With the senescence of leaves during the natural senescence,the expression of TaCLH1 s gradually decreased,and it decreased to the lowest at 20 d after flowering,and then increased.4.In the determination of chlorophyll a and chlorophyll b content and SPAD value in wheat leaves during natural senescence,we found that they were basically unchanged before the 17 th day after anthesis,they began to senescence at the 18 th day after anthesis,reached the maximum senescence rate at the 23rd-24 th day after anthesis,and stopped senescence at the 26 th day after anthesis.5.By comparing the content of chlorophyll a and chlorophyll b,SPAD value and the expression characteristics of TaCLH1 s in the process of natural senescence,we preliminarily concluded that TaCLH1 s played a less role in the degradation of chlorophyll and played a role in the later stage of leaf senescence. |
