Analysis And Gene Mapping Of Rice Premature Senescence Mutant Zs And Enhancing GABA Contents By Interference Osgaba-T1 In Rice

This study contains two parts:Analysis and gene mapping of rice premature senescence mutant zs and enhancing GABA contents by transformed OsGABA-T1 in rice.First part,it has great significance for plant normal growth and yield formation to studying the mechanism of leaves premature senescence.Leaf senescence is the final stage of leaf development,it controlled by a complex network of endogenous molecules.Organelles and macromolecular substances in cell lose their balance actively,and redistribute nutrients to developing tissues or storage organs.However,premature aging of leaves is an abnormal physiological process that seriously affects the photosynthesis of plants,which in turn influences grain filling and ultimately affects yield.In order to study the phenomenon of premature aging of rice,a rice premature decay mutant was screened from the EMS mutagenized population of the Ninggeng 1’ off population,we named as zs.In this study,we identified the mutant zs agronomic traits,chloroplast structure,and physiological characteristics,genetic analysis and gene mapping of the mutant were performed.Quantitative analysis of genes related to plastid development and chloroplast synthesis-related genes was performed.The main results are as follows:1.The main agronomic traits of the wild-type and mutant zs showed:in the light incubator at 28℃,when the mutant zs was grown for 14 d,the tip of the second leaf began to appear yellow-brown spots,with the number of growing days increased,the other leaves have tan spots.Under the conditions of normal field mana-gement,the plant height of mutant zs decreased,the panicle length decreased,and the agronomic traits such as effective tiller number,grain number per panicle,and 1000-grain weight decreased significantly.2.The scanning electron microscope results of the leaves showed that the wild-type chloroplasts were spindle-shaped and the thylakoids were arranged neatly.The chloroplasts of the mutant zs were fluffy,irregularly oval,and the thylakoid arrangement was very irregular.Peroxidation analysis of leaves showed that DAB dyes stained the mutant zs more deeply,indicating that there was accumulation of H2O2 in the leaves of the mutant zs;NBT staining was also deeper in the mutant,and there was ROS accumulation in the mutant zs leaves.3.The contents of photosynthetic pigments in wild-type and mutant zs were measured.The content of total chlorophyll in the mutant zs was decreased,the content of chlorophyll a and chlorophyll b were significantly decreased,and the content of carotenoids did not change significantly.By quantitatively analyzing the expression of genes related to plastid development and chloroplast synthesis-related genes,compared to wide-type,hydroxymethyl-backhoebhuminogen synthase(HEMC),uroporphyrinogen decarboxylase(HEME),and glutamyl-1-semialdehyde transaminase(HEML)expression is up-regulated in zs,glutamyl-tRNA reductase(HEMA),porphobilinogen synthase(HEMB),coproporphyrinogen oxidoreductase(HEMF),magnesium stilbene synthase H subunit(CHLH),The expression of other genes involved in the synthesis of chlorophyll,such as CHLD,DVR,CMD,CHLG,etc.At the same time,only ATP synthase β subunit(ATPβ)expression was significantly up-regulated in plastid development-related genes,and expression of other genes was down-regulated.4.Hybridize ’N22’ with mutant zs.F1 leaf color of the offspring was normal and F2 population was isolated.Genetic analysis showed that the phenotype of this premature decay mutant was controlled by a pair of recessive genes.By gene mapping analysis,it was located in the 600 kb interval between chromosome 12 short arm molecular markers N12-18 and ZS20.The sequencing results showed that the second exon of the cytochrome P450 monooxygenase gene.There is a single base mutation(C→T),which is a candidate gene.The second part,Gamma-aminobutyric acid(GABA)is a four-carbon,non-protein amino acid that has functions such as lowering blood pressure.In order to increase the content of GABA in rice,we used double T-DNA vectors and RNA interference technology to construct dual T-DNA interference vectors for the key GABA metabolism enzyme GABA transaminase 1(OsGABA-T1)in rice.The transgenic plants were obtained with agrobacterium-mediated method using ’Ninggeng 1’ as a receptor,and the transgenic homozygous families containing only the target fragment without hygromycin were screened by PCR and their GABA content was significantly increased.It provides a material basis for the cultivation of high levels of GABA transgenic rice with reduced blood pressure.The main results are as follows:1.Extract DNA from the leaves of T5,T6,and T7 generation transgenic plants from Zhou Lu,through PCR we amplification with the target fragment and hygromycin primers,and select the transgenic plants that contain the target band that does not contain the hygromycin marker gene.Finally,a homozygous family line not containing the hygromycin marker gene in the target band was obtained.Southern blot analysis were performed on the receptor varieties and the transgenic plants,the result showed that exogenous genes are indeed integrated into the rice genome.2.Harves for the transgenic plants of homozygous families,determine the GABA contents.The results showed that GABA contents was significantly increased in both brown rice and polished rice.The GABA contents of seeds stored for 4,8 and 12 months was determined,and the GABA contents remained basically unchanged,indicating that the storage time had no significant influence on the content of GABA.3.Real-time fluorescent quantitative PCR was used to detect the leaves of the receptor species’ Ninggeng 1 ’and the homozygous family plants.The results showed that the expression of OsGABA-T1 in transgenic plants decreased significantly,and the expression of OsGABA-T2 gene also decreased.4.The agronomic traits of receptor varieties and transgenic plants were investigated,the results showed that compared with the receptor "ninggeng 1 ",the plant height of the transgenic plants was significantly lower,and the agronomic characters such as 1000-grain weight,effective tillering number and the number of grains per panicle were decreased.