The Molecular Mechanisms Of BTAL On Regulating The Tiller Angel In Rice

The tiller angle is an important factor affecting the plant type in rice.Locating and cloning genes that regulate the angle of rice tiller and carrying out research on the molecular mechanism of rice tiller angle formation have important theoretical and practical significance for the improvement of rice plant type and the development of ideal plant type for rice super-high yield.The formation of a rice tiller angle is an extremely complicated process.In addition to genetic factors,environmental conditions(including light,gravity),hormones and other factors participate in the regulation of rice tiller angles.Previous studies have shown that the negative gravitational response of rice and the morphological changes in the cytology of tillering are the causes of changes in the angle of rice tiller.Some genes that regulate the angle of rice tiller have been cloned.However,in-depth molecular mechanisms are not yet clear.In this study,mutants with larger tiller angles and divergence growth(bigger tiller angle 1-1,bta1-1)obtained by EMS chemical mutagenesis screening were used as test materials,using map cloning,DNA recombination techniques,and agrobacterium tumefaciens.Through the techniques of genetic transformation of mature embryos in rice,fluorescence quantitative RT-PCR,and RNA high-throughput transcriptome sequencing and analysis,changes in the bifurcation angle and negative gravity response of bta1-1 mutants were observed;The BTA1 gene was cloned;the expression pattern and differential expression analysis of the gene were performed;the differentially expressed genes between bta1-1 mutant and wild type(WT)were analyzed;the enrichment pathway of differentially expressed genes was analyzed and analyzed.At the genome level,the molecular mechanism of BTA1 regulation of the rice tiller angle was analyzed.Below are the key research findings:1.The mutant bta1-1 was obtained by screening the angle of rice.Using EMS mutagenesis,we obtained a significant increase in the angle of the tiller,named bigger tiller angle 1-1(bta1-1).At different stages of rice growth,the angle of the tiller of the bta1-1 mutant was significantly higher than that of the wild type.2.The bta1-1 mutation changed the negative gravity response of rice.Through the study of the negative gravity response of WT and bta1-1 mutants,it was discovered that the negative gravity growth ability was lost of the aboveground part of the bta1-1 mutant,resulting in a greater angle of the tiller of the bta1-1 mutant.3.The bta1-1 mutation in rice also affected other agronomic traits.The results of the investigation of the agronomic trait determine that,in addition to the tiller angle,the bta1-1 mutation also resulted in changes in agronomic traits such as tiller number,stem length,effective panicle number,full-grain number,and dry matter weight.4.Clone the bta1-1 mutation site.Using a combination of map-based cloning and gene sequencing of known of controlling rice tiller angle,it was discovered that there is a mutation from G to T in the third exon of the known LA1 gene,by changing the codon from GAG to TAG and by encoding glutamic acid(Glutamic acid,Glu)into a stop codon,leading to the premature termination of translation.5.The BTA1 gene can complement the bta1-1 phenotype.The plant expression vector was constructed 35S:BTA1/LA1-GFP and the constructed vector was transformed into the bta1-1 mutant to obtain an independent transgenic strain.The results of phenotypic observations indicate that the BTA1/LA1 gene can integrate the phenotype with a larger tiller angle of the bta1-1 mutant,indicating that the G to T mutation of the LA1 gene is the cause of the bta1-1 phenotype.suggesting that bta1-1 is an allelic mutation of lazy1.6.The results of the bioinformatic analysis showed that 40 proteins similar to BTA1/LA1 in 17 species contain the typical EAR motif,indicating that the protein similar to BTA1/LA1 as an active suppressor negatively regulates the expression of the response genes to stress.The results of evolutionary analysis and collinearity analysis showed that proteins similar to BTA1/LA1 in monocotyledon plants are more closely related.7.The BTA1/LA1 gene is expressed in different tissues and organs at different stages of growth and development.The real-time quantitative RT-PCR results showed that BTA1/LA1 was expressed throughout the growth period and in various rice organs.8.The results of real-time quantitative RT-PCR showed that compared with WT,BTA1 /LA1 expression was significantly reduced in all stages and portions of bta1-1 mutation,suggesting that bta1-1 mutation inhibits BTA1/LA1 expression.9.The bta1-1 mutation changed the subcellular localization of the BTA1/LA1 protein.The results of subcellular localization analysis and plasma wall separation analysis indicate that the bta1-1 mutation simultaneously changes the subcellular localization pattern of the BTA1/LA1 localization in the cell membrane and nucleus.The partial protein BTA1/LA1△C160 after the bta1-1 mutation lost its nuclear localization and was localized only in the cell membrane.10.The bta1-1 mutation changes the expression of auxin signal transduction and genes related to polar auxin transport.Compared to WT,the expressions for Os PIN1,Os PIN5 b,Os ABCB14,Os CLIP,and Os IAA6 were significantly down-regulated,while those for Os PIN2,Os AUX1,and Os COLE1 were significantly down-regulated.11.The bta1-1 mutation changes the expression of multiple pathways and genes with different biological functions.High-throughput RNA sequencing results showed that the bta1-1 mutation significantly changed the expression of 1025 genes.These differentially expressed genes are highly enriched for auxin signaling and other pathways.BTA1/LA1 can participate in the assembly of nucleosomes and chromatin by mediating protein-DNA interaction.These indicate that BTA1/LA1 controls the tiller angle of the rice by regulating the transport of the auxins and the transduction of the auxins signal,resulting in an asymmetrical distribution of the auxins.In summary,this study shows that bta1-1 is a lazy1 allelic mutation.The loss of the nuclear localization signal and the EAR motif leads to the loss of the LA1 function.The bta1-1 mutation inhibits the expression of LA1,it shows that the nuclear positioning signal and the EAR motif are particularly important for the LA1 function.At the same time,the expressions Os PIN1,Os PIN5 b,Os ABCB14,Os CLIP,and Os IAA6 were down-regulated and the expressions Os PIN2,Os AUX1,and Os COLE1 were up-regulated,indicating that BTA1/LA1 influences the negative gravity of the rice by adjusting the auxin signal transduction and polar transport,and finally influence tiller angle.