| Wheat(Triticum aestivum L.)is one of the world’s most important food crops.With climate change,various harsh environmental factors severely constrain wheat production.To ensure food security,it is urgent to develop new varieties featured with improved tolerance to abiotic stress,stable yield and wide adaptation.Mining and utilization of elite germplasm and gene resources is the most economical and effective way to resolve this challenge.Our previous studies showed that TaNAC67 was involved in the response to a variety of stresses,and its overexpression resulted in enhanced tolerance to multiple abiotic stresses in Arabidopsis.However,the undelined mechanisms are largely unknown,and its roles in regulation of the development for major agronomic traits are also needed to elucidate.To this end,TaNAC67 was transferred into rice and a serial of experiments were performed,including cis-acting element prediction in promoter regions,expression pattern analysis,transgenic rice phenotyping,transcriptome analysis,yeast two hybrid assay,DNA polymorphism analysis,functional marker development and association analysis.The main ressults are as follows:(1)The wheat TaNAC67-6A,-6B and-6D genes were cloned from common wheat Hanxuan10.Gene structure assay showed that TaNAC67s contained only one exon without intron.Cis-acting element assays revealed the presence of a variety of abiotic stress responsive elements in the upstream promoter regions,however,the number of cis-acting elements and their distributions differed remarkably between different genes.(2)Gene expression pattern assays revealed that TaNAC67-6A,-6B and-6D were induced by salt,drought,abscisic acid and low temperature stress,while depressed by high temperature,and different TaNAC67 members had different expression patterns under same conditions.The order of sensitivity for TaNAC67s was low temperature>ABA>high salinity>drought>heat.The expression levels of TaNAC67-6B and-6D were generally higher than that of TaNAC67-6A,and the expression levels of the same gene in leaves were higher telative to roots.(3)The phenotyping results showed TaNAC67 overexpressing rice lines were improved in several traits relative to wild type control,including ear length,spike number,number of primary and secondary branch,strile number of panicle,effective tiller number,grain length and grain width.However,the plant height of transgenics was significantly reduced cpmpare to that of WT.(4)Under drought stress,the survival rate of transgenic rice was about 25-40%,and that of wild type was less than 10%,suggesting the transgenics acquired enhanced tolerance to drought.Under salt stress,the number of roots,cell membrane stability,SPAD value and survival rate of TaNAC67 overexpressing lines were significantly higher than WT,and the osmotic potential and Na~+content were significantly lower than WT control,indicating that TaNAC67 transgenic rice had enhaced tolerances to salt.(5)The transcriptome assays showed that 72 and 55 genes were differentially expressed under PEG and NaCl stresses,respectively,of which 54 and 17 genes were up-regulated,and 18 and 38 genes were remarkably down-regulated relative to the WT control.The differentially expressed genes were mainly related to drought stress,while fewer genes were identified under salt stress.The transcriptome assay results were further valaidated by qRT-PCR assays.The genes upregulated in transgenic lines under drought stress mainly involved glutathione,glucose and nitrogen metabolisms.(6)DNA polymorphism assays showed that there was a SNP and a 126-bp InDel in the promoter region of TaNAC67-6A,and two SNPs in the promoter region of TaNAC67-6B.For TaNAC67-6D,one SNP was located in the promoter region and one in the coding region.Based on the identified SNPs,functional markers SNP-A-1,SNP-A-2,SNP-B-1,SNP-B-2,SNP-D-1 and SNP-D-2 were developed accordingly.(7)Association assays showed there was no significant correlation between the functional markers of TaNAC67-6A and-6B and the observed agronomic traits.However,the two functional markers of TaNAC67-6D,SNP-D-1 and SNP-D-2,were significantly associated with panicle length and spikelet number per spike,respectively.It was consistent with the phenotyping results of transgenic rice.Among the natural populations of wheat,there were three major haplotypes in TaNAC67-6D,among which Hap-6D-3 was an elite haplotype for spike length and spikelet number per spike and it has been positively selected in wheat breeding in China. |
