| Poaceae crops(such as rice,wheat,sorghum,etc.)have a short post-maturity period.If they are not harvested in time at the later stage of maturity or encounter many days of overcast rain in the middle and later stages of grain filling,seeds will be induced to germinate on the ears.This phenomenon is called pre-harvest sprouting(PHS).The essence of PHS is that the balance of seed dormancy is broken.The rainy and humid environment will affect the dormancy of crops such as rice,resulting in an increased probability of PHS.There is a direct link between the starch metabolism process and seed germination,but past research often focused on the seed germination in the process of starch hydrolysis effects on seed germination,the key genes and seed germination in the process of starch synthesis,especially the relationship between panicle germination in the process of seed maturity almost few studies and reports.Strarch branching enzyme(SBE)is one of the key enzymes involved in strarch synthesis,which is the only branch in traduced in the process of strarch synthesis enzyme.This study uses rice as material to carry out crop PHS research,which mainly includes the following two parts.One is to carry out high-humidity treatment of rice seeds in the late stage of maturity,to explore the dynamic expression patterns of seed dormancy-related genes under high-humidity environment(simulating continuous rainy weather),and to provide Provide the basis and suitable marker genes for the molecular mechanism research of environmental factor-induced crop PHS;Second,the OsSBE3 and OsSBE4 RNAi double-interference materials of rice starch branching enzyme gene(OsSBE)genes were obtained in the early stage of this experiment.The study found that the OsSBE3/4 double-interference materials had obvious PHS phenotype.Based on this material,this thesis uses molecular biology and biochemical methods to study the molecular mechanism of the two most important starch branching enzymes in rice seeds,OsSBE3 and OsSBE4,involved in regulating of PHS,and the following results are obtained:1、In order to explore the molecular mechanism of continuous rainy weather affecting seed dormancy in the late stage of rice seed maturation.We used artificial climate chamber cultivation and regular water spray to simulate continuous rainy conditions to deal with japonica rice at 26,28,30,and 32 days after pollination(DAP),and detected the germination on the ear.The results showed that the seed maturity was is an important factor in the production of PHS induced by high humidity.The japonica rice Dongjin before DAP28 rarely germinated after high-humidity treatment,and the seeds of DAP30 and above mostly germinated after high-humidity treatment.2、Subsequently,the test material of the rice crop at DAP30 was treated with high humidity,and the synthesis of hormones ABA and GA,and the related genes of their signal transduction pathways were detected at different time points after the high humidity treatment and the dynamic expression of α-starch hydrolase-related genes in early seed germination.The results showed that high-humidity treatment could rapidly induce the expression of ABA degradation genes OsABA8ox1 and OsABA8ox3 and GA synthesis genes OsGA3ox2 and OsGA20ox1,while inhibiting the expression of key ABA synthesis genes OsNECD1,OsNECD2 and OsNECD3;The expression levels of α-starch hydrolase genes OsAmy1 A,OsAmy3 B,OsAmy3 C and OsAmy3 D were also rapidly up-regulated after water spraying for 72 h,and the hydrolyzed starch provided material and energy for seed germination.The results of this study will provide a basis for analyzing the molecular mechanism of PHS induced by rice in the mature stage under the rainy and high humidity environment.3、The laboratory obtained OsSBE3/4 RNAi transgenic lines in the early stage,and the transgenic material was found to have smaller,chalky and significantly shrunken grains and a distinct PHS phenotype compared to Nipponbare(hereinafter referred to as NIP)controls under field planting conditions.Germination experiment of freshly harvested seeds at different developmental stages showed that OsSBE3/4RNAi seeds germinated 25 days after pollination(DAP25)while NIP hardly germinated.Consistent with past studies,the wild type requires a certain post-ripening time to achieve a high germination rate.4、Then,when we performed physiological and biochemical tests on soluble sugars such as sucrose,fructose,and glucose in OsSBE3/4 RNAi materials and NIP seeds at different developmental stages,we found that the content of soluble sugars in OsSBE3/4 RNAi seeds increased significantly.At the same time,according to the marker genes in the PHS process screened out in the previous part of this paper,their expression changes in OsSBE3/4 RNAi materials and NIP were detected.The results showed that the expression levels of GA synthesis genes(OsGA20ox1,OsGA20ox2,OsGA3ox2),ABA hydrolysis genes(OsABA8ox3)and starch hydrolase genes(OsAmy1A)in OsSBE3/4 RNAi seeds were significantly higher than those in NIP seeds at different stages of seed development.Therefore,we speculate that the down-regulated expression of the starch synthase gene OsSBE in OsSBE3/4 RNAi seeds leads to blocked starch synthesis and increased soluble sugar content.Sugar signaling affects the synthesis of GA and ABA by promoting the expression of genes related to GA synthesis and ABA hydrolysis,resulting in increased OsAmy1 A expression,which further promotes the hydrolysis of stored substances,resulting in the PHS phenotype.In conclusion,our research results will help us to understand the molecular mechanism of rice PHS production induced by environmental factors(high humidity),and provide good spatiotemporal expression marker genes for our follow-up PHS research.At the same time,OsSBE3/4 double-interfering plants provided us with good genetic material to study the gene defect of starch synthesis and seed dormancy disorder in grain-filled seeds,as well as the internal relationship and molecular mechanism of PHS production.This study can enrich the regulatory network of PHS,and the results will help us provide new ideas for improving the dormancy of crops and solving the problem of PHS in the future. |
