Isolation,functional Analysis And Application Of Tissue-specific Promoters In Wheat

Wheat(Triticum aestivum L.)is the world's second largest food crop,wheat grain is the kind of the most important staple food for human beings,and it provides more than 35% of the world's population with calories and proteins.Isolation and characterization of plant tissue-specific genes and their promoters are very important.It not only helps us to predict the gene functions and understand the gene regulation during plant growth and development,it also can be utilized as powerful tool in genetic engineering research as well.But the study of tissue-specific genes and promoters of monocotyledons,especially in wheat,is far from enough and has lagged behind other plants,such as rice.In the present study,the wheat tissue-specific genes and their promoters were analyzed and applied in genetic engineering for construction of male sterility lines and the non-destruction visual screening of transgenic lines in wheat.In addition,a pollen-specific promoter Os PSG076 from rice was isolated and confirmed in transgenic rice.The main results are as follows:(1)Public wheat microarray data of seven organs including coleoptile,root,leaf,pistil,anther,embryo,and endosperm from NCBI database were downloaded for analyses and 604 probesets representing genes showed tissue-expressed specificity.In addition,a total of 330 genes were successfully located on wheat chromosomes.Interestingly,the genes representing the same tissue-specificity were always clustered on chromosomes,and some genes were found to have multicopy features as located on the different chromosomes.(2)RT-sq PCR and RT-q PCR analyzed results showed that the expression patterns of predicted 36 genes were accordance with microarray data.To further analyze the upstream promoters,vectors of p Col1::GUS,p Root2::GUS and p Leaf1::GUS were constructed.Agrobacterium-mediated transformation of transient expression assay in wheat coleoptile showed that the Ta Col1 promoter could drive the gus reporter gene expressed in wheat coleoptiles.Vectors p Root2::GUS and p Leaf1::GUS were also transformed into Arabidopsis by Agrobacterium-mediated transformation method,GUS histochemical staining results showed that the Ta Root2 represents root-specific regulation pattern in Arabidopsis while Ta Leaf1 is a leaf-preferential promoter in Arabidopsis.(3)With the aim to construct a wheat non-destruction visual screening method for transgenic lines,vectors p Root7::Ds Red and 1Dx5::Ds Red were constructed and Ta Root7 was the putative wheat root-specific promoter from our analysis and 1Dx5 promoter was previously reported to be endosperm-specific.Transgenic wheat plants harboring p Root7::Ds Red or 1Dx5::Ds Red were obtained by particle bombardment technique and were analyzed.(4)With the aim of construction of wheat male sterile lines,vector Ta PSG076::Barnase(p14B)was constructed with the pollen-specific promoter Ta PSG076(1.4 kb)which was cloned and identified previously in our laboratory.Transgenic wheat lines harboring p14 B were obtained by particle bombardment method and were analyzed.(5)Upstream promoter of a wheat Ta PSG076 homologous gene,designated Os PSG076,was isolated and identified from rice,and the expression vector Os PSG076::GUSplus was constructed with 987 bp length of Os PSG076 promoter and transformed into rice by Agrobacterium-mediated transformation method.GUS histochemical staining showed that the Os PSG076 promoter could drive reporter gene gus expressed in rice pollen exclusively with high efficiency and intensity.(6)Overexpression(OE),RNA interference(RNAi),and gene editing(CRISPR/Cas9)expression vectors for gene Os PSG076 functional verification were constructed and the corresponding rice positive transformants were obtained.In brief,tissue-specific genes from seven tissues of wheat were analyzed.Results thus obtained could help us understanding the functions of related genes and also provide the resource for wheat tissue-specific promoter exploitation.Upstream promoters of three candidate genes were functionally validated in planta,and the application of tissue-specific promoters in wheat was also studied.In addition,the pollen-specificity of Os PSG076 promoter was verified in transgenic rice,and rice transformants of expression vectors for functional verification of Os PSG076 gene were also obtained.These results confirm the feasibility of the high-through tissue-specific promoter screening method and the potential application value of the tissue-specific promoters.