| Sorghum(Sorghum bicolor L.Moench),which has high stress resistance and extensive use,is an important food crop with broad application prospects and a kind of energy crop.Tillering and leaf are important organs that could affect grain yield and plant architecture in crop.Therefore,identification and functional analysis of these genes will be helpful for the understanding of genetic mechanism of these two traits and breeding of high-quality varieties of sorghum.Two mutants selected from EMS mutation population of BTx623 are used for this study.One is non-dormant axillary bud 1(nab1),a mutant with increased tillers and reduced plant height,and the other one is narrow leaf and dwarf 1(nld1),a mutant with narrow leaf,dwarf and low fertility.Using the map-based cloning method,we isolated the NAB1 and NLD1 genes from sorghum.Meanwhile,we analyzed their molecular functions.The main results were as follows:(1)NAB1 was mapped on the chromosome 6 and further delimited within a 50-kb region by using an F2 population,which was derived from a cross between nab1 mutant and ShangZhuang broomcom.Comparing the genomic sequence of the mapping region(?50-kb)between the BTx623 and nab1 mutant,only a single nucleotide change,C to T,at positon+223 of Sobic.006G170300 was found,which led to a premature stop codon in nab1 mutant.Homologous sequence analysis showed that NAB1 was a single copy gene in sorghum and encoded a carotenoid-cleavage dioxygenase7(CCD7)protein,which was orthologous to rice HTD1/D17 and Arabidopsis MAX3.Subcellular localization showed that NAB1 protein was localized specifically in chloroplasts.Quantitative reverse transcription PCR analysis showed that NAB1 expressed in all tested tissues and stronger in the tiller base and node tissues.The exogenous application of GR24 can completely suppress the tillering of nab1 mutant,the root exudates of nab1 seedlings failed to promote the germination of Orobanche seeds and the content of strigolactone was undetectab1e in nab1 mutant revealed that NAB1 participated in the biosynthetic process of strigolactones.Meanwhile,polar auxin transport(PAT)assay showed that nab1 mutant plants has stronger PAT;RNA-seq data showed that a lot of differentially expressed genes(DEGs)were existed in nab1 mutant and IAA-related genes were abundant among the DEGs,what's more is that all IAA-transport-related genes were up-regulated among the IAA-related genes.All of these results suggested that NAB1 is related to the biosynthesis of strigolactones,regulating sorghum shoot branching by interacting with auxin transport.(2)NLD1 was mapped on the chromosome 2 and further delimited within a 22-kb region by using an F2 population,which was derived from a cross between nld1 mutant and ShangZhuang broomcorn.Comparing the genomic sequence of the mapping region(?22-kb)between BTx623 and nld1 mutant,only a single nucleotide change.C to T.at positon+575 of 'A' gene was found.which led to the transformation of amino acid at 192.from "Pro" to "Leu",in nld1 mutant.The rice transgenic plants(Cas9,ZH11),which have knocked down the OsNLD1 gene,showed similar phenotypes with nld1 mutant;Quantitative reverse transcription PCR analysis showed that NLD1 expressed in all tested tissues and stronger in leaf tissues;Functional analysis revealed that NLD1 participated in the biosynthesis of shikimate;the content of tryptophan and IAA in nld1 mutant plants were both less than those of wild-type;Meanwhile,different concentrations of tryptophan can regain the lateral root phenotype of hld1 mutant seedlings.RNA-seq data showed that a lot of differentially expressed genes(DEGs)were existed in nld1 mutant,including many tryptophan-related and IAA-related genes.All of these results suggested that NLD1 modulated the growth of sorghum by participating in the biosynthesis of shikimate,which can influence the content of IAA. |
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