Mining Of Genes Related To Leaf Color Yellowing In Tobacco Yellow Leaf Mutants Based On Transcriptome Sequencing

Tobacco is an important economic crop with leaves as the yield organ.During the mature process of tobacco leaves,chlorophyll will degrade and the leaf color "turns yellow".Tobacco leaves are layered from bottom to top,and the characteristics of "yellowing" in slices make the leaves of tobacco plants need to be harvested in layers and multiple times,which has become a bottleneck for saving labor and realizing mechanized harvesting.In the early stage,the research group discovered a yellow mutant plant in the DH line of anther culture.The mutant was yellow-green throughout the growth period,and had the effect of promoting the concentrated maturation of tobacco leaves and improving the quality of tobacco leaves.The gene remains to be discovered,and the mechanism related to leaf color yellowing remains to be elucidated.Therefore,in this study,the yellow leaf mutant,yellow leaf K326 and K326 were used as test materials to investigate the agronomic properties of the materials,and to clarify the appearance differences between the materials;Differences in photosynthetic pigment content and chlorophyll synthesis blocking steps during growth period;chloroplast ultrastructural damage was observed by transmission electron microscopy;key genes related to leaf color yellowing were screened by transcriptome sequencing technology.The mechanism of leaf color yellowing of the mutant was expounded,which provided a theoretical basis for breeding and utilizing concentrated mature varieties and cloning mutant genes.The main findings are as follows:(1)The phenotype observation of the test materials showed that the yellow-leaf mutant and the yellow-leaf K326 were yellow-green throughout the growth period,and the yellow-leaf K326 was similar to K326 in appearance.By measuring agronomic traits,it was found that compared with the yellow leaf mutant,the yellow leaf K326 obtained by backcrossing was not significantly different from K326 in terms of plant height,leaf length,leaf width and number of leaves after budding,indicating that the yellow leaf K326 It is closer to K326 in appearance,and the genetic background of the two is similar.(2)The measurement results of photosynthetic pigments and chlorophyll precursors showed that the chlorophyll content of the two yellow leaf materials at each growth stage was significantly lower than that of K326,and the chlorophyll change trend of the three materials in each growth stage was the same.The difference in chlorophyll content between the leaf material and K326 reached the largest,and the total chlorophyll content of the yellow leaf mutant and the yellow leaf K326 was58.57% and 52.07% of that of K326,respectively.The results of the determination of chlorophyll synthesis precursors showed that the hindered step of chlorophyll synthesis was the biosynthesis of coproporphyrin Ⅲ(Coprogen Ⅲ)to protoporphyrin Ⅸ(Proto Ⅸ).(3)Electron microscope observation showed that compared with K326,the distribution of chloroplasts in the mesophyll cells of the two yellow leaf materials was irregular,the boundary was unclear,the shape of chloroplasts was irregular,some chloroplast membranes were damaged,the number of grana and the number of inner thylakoid layers were obvious.Decreased,the lamellae are loose and scattered,the arrangement and distribution are irregular,and the structure is fuzzy.It indicated that the chloroplast development of the yellow-leaf mutant and the yellow-leaf K326 was blocked and the structure was damaged.(4)Transcriptome analysis showed that there were 935 DEGs between the two yellow leaf materials and K326,and these DEGs were mainly enriched in copal-8-ol diphosphate 3-catabolism,cis-diphenol metabolism,magnesium chelation The synthase activity,chloroplast m RNA processing,porphyrin and chlorophyll metabolism and other processes indicated that the yellowing of the mutant’s leaf color was closely related to these metabolic processes.Combined with physiological indicators and subcellular structure observation results,we further screened key genes and obtained 24 genes related to leaf color.Among them,CHLH and CHLD genes were hardly expressed in the two yellow leaf materials,which led to the block of chlorophyll synthesis and the decrease of chlorophyll content;The down-expression of EMB2654,PGR3,GLB1,FAX1,WAXY,NHX7 and other genes in the two yellow leaf materials led to the blockade of transcription,translation,thylakoid development and amyloplast formation in chloroplast development,resulting in chloroplast dysplasia and structural damage.Based on the results of physiological,subcellular structure and transcriptome data analysis,it is speculated that the yellow leaf mutant gene may affect the expression of CHLD,CHLH,EMB2654,PGR3,GLB1,FAX1,WAXY,NHX7 and other genes,resulting in poor chloroplast development and blocked chlorophyll synthesis,the chlorophyll content decreases,eventually causing the leaves to turn yellow.