Cloning And Functional Analysis Of HTD12 Regulating Plant Height And Tiller N Umber Of Rice(Oryza Sativa L.)

It is well known that to develop the elite variety with ideal plant architecture is one of the best ways to increase grian yield of rice.In generally,the plant architecture of rice is consist of plant height,tiller traits(including number and angle),leaf and panicle morphology,Plant height and tiller number determinate the capability of lodging-resistance and number of effective panicles per plant,respectively.To identify the genes controlling plant height and tiller number can not only offer basis for elucidating the genetic network of plant height and tiller number,but also provide favorable genes for improving the plant architecture of rice.In this study,HTD12 gene regulating plant height and tiller number was characterized using a high-tillering and dwarf mutant htd12 with map-based cloning methods.The main results are as follows:1.Compared with the wild type YIL18,the plant height of htd12 was decreased and the tiller number was increased.At the mature stage,plant height of htd12 was 56 cm,significantly shorter than that of YIL18(79 cm),while number of effective panicle of htd12was 78,being four times as much as that of YIL18.Moreover,htd12 also displayed shorter panicle and less grain number.Compared with YIL18,the length,number of primary branch and grain number per main panicle of htd12 decreased by 29%,52%and 58%,respectively.2.Genetic analysis using an F2 population derived from the cross between htd12 and a japonica variety Nipponbare showed that the segregateion rate of the YIL18 and htd12 mutant plants fitted a 3:1 ratio,indicating that the htd12 phenotype was controlled by a single recessive nucleus gene.Using an F2 population derived from the cross between htd12 and a japonica variety Zhonghua 17,HTD12 was limited to a 1300-kb region adjacent to the centromere on the short arm of chromosome 12.Expession profiling using an Affymetrix whole-genome array and real-time quantitative PCR assays showed that two predicated genes in the HTD12 mapping region exhibited a significant expression change.Finally,one single nucleotide change,G to A,at the end of the first intron of LOC_Os12g21710,leading to failure of the second exon in the htdl2 transcription was detected in the mutant.3.To verify the SNP happened in LOC_Os12g21710 was responsible for the phenotype of htd12,a pRNAi construct was introduced into YIL18 and Teqing,respectively.Compared with control,all the independent RNAi transgenic lines were characterized as high-tillering and dwarf,as well as weaken main panicle.These results demonstrated that LOC_Os12g21710 is HTD12,regulating plant height and tiller number in rice.4.Expression pattern analysis showed that HTD12 was detected in almost all tissues,especially with high levels in leaves,nodes and panicles.The expression levels of HTD12 were down-regulated in htd12 comparing with those in wild type YIL18.Moreover,the expression signals of RNA in situ hybridization were strongly detected in the mesophyll,axillary bud and immature spikelet.Transient expression of the construct p35S::HTD12-GFP showed that GFP-HTD12 was localized in the chloroplast,demonstating that HTD12 is a chloroplast-protein.5.Multiple sequence alignment showed that HTD12 was an orthologous gene of Z-ISO,which encoded a carotenoid isomerase involved in carotenoid biosynthesis.Comparsion of protein structure between HTD12 and htd12 showed that the SNP in the htd12 mutant generated 49 amino acid residues deletion which disturbed the original structure with greatly reassociation.Furthermore,a critical residue histidine 151 related to the activity of isomerase missed,resulting in obstructing synthesis of carotenoid.6.Detection of carotenoid content showed that carotenoid content in htd12 ws decreased compared with the wild type YIL18.Further detection of strigolactones(SLs)concentration in endogenesis and root exudates showed that the levels of 2’-epi-5DS,a native SL of rice,in htd12 were significant lower than those in YIL18,suggesting the mutation of htd12 led to disturbing carotenogenesis,reducing content of the carotenoid and SLs,and lower level of SLs caused increase of tiller number and decrease of plant height in the htd12 mutant.