Cloning And Functional Analysis Of A Narrow Leaf Gene NAL13 In Rice

Leaf is the main place of rice for photosynthesis,respiration and transpiration.Leaf morphology is related to various physiological functions of rice.By improving the morphology of rice leaves,increasing the leaf area index of rice,and improving the photosynthetic efficiency of the population,thereby the yield and quality of rice can be improved.In this study,a mutant nal13 with narrow leaf phenotype obtained from a big mutant library,which caused by the γ-ray mutagenesis treat to Zhonghua11 varatiey.In this study,a series of experimental methods like morphology,physiology and cytology were used to explore the causes of phenotypic changes such as leaf narrowing,increased tillering,lower plant height,and thinner stems.The NAL13 gene was mapped using map-based cloning technology,and the mutation site of NAL13 was found by sequencing.On this basis,related experiments such as functional complementarity,subcellular localization and tissue expression analysis of this gene were carried out,which laid the foundation for functional analysis of NAL13.At the same time,it also provides new important genetic resources for elucidating the molecular mechanism of leaf growth and development and molecular breeding of ideal plant types in rice.The detailed research results are shown as follows:(1)Phenotype analysis of nal13: Compared with the wild type ZH11,the mutant nal13 showed a narrower leaf phenotype at the seedling stage;at the tillering stage,in addition to the narrowing of the leaves,with the increase of tillers,the decrease of plant heightand thinning of the stem.Through dynamic observation of nal13 tillers,it was found that the increase of nal13 tillers was mainly the tillers formed by high axillary buds in the later period,and finally the number of tillers of nal13 could reach2-3 times that of wild type;at the same time,each stem node became thinner,and the statistics of the number of vascular bundles in the next section showed that the number of vascular bundles decreased significantly.The nal13 has an earlier heading stage,which is about 20 days earlier than wild type.At the maturity stage,plant height,panicle length,primary branche number and secondary branche number of nal13 decreased significantly,however,the number of effective panicles increased,and the total number of seeds per plant increased significantly.(2)Histological analysis of nal13: The leaves of mutant nal13 became narrower.Microscopic observations showed that the number of large veins and small veins of mutant nal13 was dramatically reduced.Paraffin section results showed that the number of vesicular cells in the large vascular bundles of nal13 leaves decreased,and the size and number of vesicular cells between the small vascular bundles became smaller,making the distance between the small veins closer.Using scanning electron microscope and transmission electron microscope to observe the leaves,it was found that the size and number of stomata of the mutant nal13 leaves were not significantly different,but the number of silicon papillae around the stomata decreased.The arrangement of chloroplast thylakoids in mesophyll cells was not significantly different,but the number of osmium particles increased.(3)Analysis of nal13’s response to hormones: Compared with the wild type,the auxin content of the mutant nal13 in the seedling stage and tillering stage was remarkably decreased,and the tillering stage was more obvious.Therefore,the expression of genes related to the auxin synthesis pathway at the tillering stage was found that the expression of the auxin synthesis genes YUCCA1,YUCCA2,YUCCA7,YUCCA8,and YUCCA9 in the mutant nal13 largely decreased,and the expression of auxin receptor TAR3 decreased.The result of treatment of wild-type and mutant nal13 seedlings with exogenous NAA showed that low-concentration NAA promoted the growth of mutant nal13,and high-concentration inhibited the growth of mutant nal13.(4)Genetic analysis and fine positioning of nal13: Genetic analysis showed that the narrow-leaf mutant nal13 was controlled by a single recessive nuclear gene,and NAL13 was located between the Indel markers M4 and M5 of rice chromosome 7using a map-based cloning method with a physical distance of 150 kb.Through sequencing analysis,it was found that NAL13 was mutated.The 313 nucleotide C in the coding region was mutated to T,which caused the encoded amino acid to change from glutamine to a stop codon,and the termination of translation was advanced.The gene encodes a cytochrome P450.This gene has not been previously reported,suggesting that NAL13 is a new narrow leaf gene.(5)The genetic complement experiment of nal13: In order to confirm that the NAL13 mutation is the reason for the appearance of the mutant nal13 phenotype,a complementary vector was constructed and transferred into callus formed by nal13,which has now reached the differentiation stage.Later,the NAL13 gene knockout CRISPR / Cas9 vector will be used for verification.(6)Analysis of the expression pattern of nal13: By detecting the expression level of the narrow leaf gene NAL13 in the leaves,leaf sheath,culm,panicle and tiller buds,it was found that the expression level of NAL13 gene was highest in leaves,followed by tiller buds and third in panicles,and the expression of NAL13 in nal13 was generally higher than ZH11.In addition,by protoplast transformation and localization analysis,the protein of NAL13 was localized on the nucleus and plasma membrane.