Cloning And Analyses Of Genes Involved In Response To Drought Stress In Maize

Maize(Zea mays L.)is not only an important economic crop in the world,but also a vital resource for forage and food industry.In China,the yield of maize is higher than those of the other crops except rice and plays an important role in the national economy.Water-deficit stress is one of the major environmental stresses that adversely affect plant growth and crop yield worldwide.In our country,about 60%of the maize area is under water-deficit stress,which causes 20%-30%of reduction in yield every year and affects the development of national economy and the normal life of people in the arid field.Periods of water deficit in soil could occur at any time during the crop season,but maize is particularly sensitive to water stress around flowering time,which cause remarkable yield loss.Heading time,which is just before tassel flowering,is one of the most important stages when maize productivity would be affected severely if plants encounter stresses.Understanding the mechanism of plant tolerance to environmental stresses and the molecular basis of plant responses to water stress,especially at the crucial development stage might provide new strategies to improve the stress tolerance of important agricultural plants.To advance our understanding of maize plants response to drought stress at heading stage,we adopted a genomic approach to monitor the transcriptome change of maize leaves subjected to 1 day and 7 days of water deficiency stress,using the Arizona 57K oligo microarray slides,and a large number of genes in response to water deficit were found.And two subtractive cDNA libraries of maize leaves at heading stage under 1 day and 7 days water stress conditions were constructed by adopting suppression subtractive hybridization(SSH)method.Further more,the full-length cDNAs of maize AtFtsH2-like and typeâ…¡H⁺-PPase genes were isolated by RT-PCR approach coupled with in silico cloning with the ESTs obtained from SSH libraries as the probes,and the basic analyses of these genes were performed. The main results were shown as follows:In this study,maize plants at heading stage suffered from 1 day(short-term)and 7 days(long-term)water-deficit stress separately and the gene expression profiles of their leaves were examined using an Arizona Maize 57K Oligonucleotide Array representing more than 30,000 identifiable unique maize genes.The genes of the stressed sample which were more two-fold than that of the control were defined as stress-inducible(up-regulated)genes.Similarly,the genes expressed in the stressed samples at less than half of the control values were regarded as stress-repressible (down-regulated)genes.We identified 102(52%of differential expression genes)and 93 genes which were stress-inducible and repressible in the short-term stress experiment,while 332(33%of differential expression genes)and 676 genes were upand down-regulated in the long-term stress experiment.And there were only 22 genes whose expressions were induced and 37 genes that were repressed in common under both treatments.The results showed that gene expression changes were significantly different in the short-term and long-term water-deficit treatments.The genes with differential changes in expression by the 1d and 7d water-deficit stress can be divided into several function categories according to the functional categories of the Arabidopsis proteins.It was noteworthy that about one third of 1d stress-induced genes with known or putative functions participated in various cellular signaling pathways,indicating that signal transduction related genes played important roles in the early-responses to water stress of maize leaves.The 7d stress regulated genes were involved in a broad range of cellular and biochemical activities.The most notable genes may function in compatible osmolyte metabolism,particularly in proline, sucrose,trehalose and raffinose metabolism in the leaves.For example,the genes encodingΔ1-pyrroline-5-carboxylate synthetase(P5CS)andΔ1-pyrroline 5-carboxylate reductase(P5CR)were induced and a putative proline dehydrogenase (ProDH)gene was repressed in leaves following long-term water stress.And it is noteworthy that several genes encoding key enzymes functioning in raffinose metabolism were up-regulated by water stress in maize leaves.By contrast,both a putative trehalose-6-phosphate synthase(TPS)gene and a putative trehalose-phosphatase(TPP)gene for the biosynthesis of trehalose were significantly down-regulated in leaves under water-deficit condition.However,a putative trehalase gene involved in the trehalose degradative pathway was up-regulated by the stress. These results revealed that the metabolism related genes differentially expressed in controls and samples undergone water-deficit stress were prominent in the leaves of plants at heading stage.We have submitted the microarray data to public database and now all expression data are available at the NCBI Gene Expression Omnibus(GEO) database(http://www.ncbi.nlm.nih./gov/geo/)under the GSE series accession number GSE10596.In order to identify genes induced during the drought stress response in heading maize(Zea mays),suppression subtractive hybridization(SSH)was performed using cDNAs prepared from maize leaves at heading stage treated with water-deficit stress for 1 day and 7days as testers and cDNAs from the control maize leaves as drivers. Two subtractive cDNA libraries were constructed,in which the rate of recombination was 95%and the size of inserts ranged from 300 bp to 600 bp.Analysis of sequences from the positive clones picked randomly revealed that many drought stress associated genes,including dehydrin,sucrose synthase 3,betaine aldehyde dehydrogenase,DRE binding factor 1,et al.,were obtained.The successful construction of the two subtracted cDNA libraries enable us to identify new differentially expressed genes involved in the resistance mechanism of heading maize plants.A 192 bp EST was obtained by screening our successful constructed SSH-cDNA library of maize leaf at the heading stage.Following similarity searching from BLASTN at NCBI,the partial cDNA of putative FtsH protease gene fragment was identified.By in silico cloning analysis using the 192 bp EST as a query probe,two contig sequences,2510 and 2430 bp in size,were obtained.And then two maize cDNAs with complete open reading frame(ORF)encoding FtsH protease proteins were amplified by RT-PCR using two pairs of gene-specific primers designed based on the results of in silico cloning.The two cDNAs sequences were 95%identity to each other on nucleotide acid level,and the encoded proteions had 97%sequence identity and showed high degree of amino acid sequence similarity with FtsH2 protease of Arabidopsis thaliana.The full-length cDNA sequences were designated as ZmFtsH2A and ZmFtsH2B,and deposited in the GenBank database under the accession numbers EU257690 and EU257691,respectively.ZmFtsH2A(2510 bp) consisted of an open reading frame(ORF)of 2304bp,a 5'untranslated region(UTR) of 241 bp and a 3'untranslated region of 217 bp.Similarly,ZmFtsH2B(2430 bp) composed of a 2034 bp ORF,a 222 bp 5'UTR and a 156 bp 3'UTR.Both the ORFs of two genes encoded 677 amino acids containing the putative AAA domain and the Zn²⁺-binding motif,which were the characteristic of FtsH metalloproteases family. The genomic sequences corresponding to ZmFtsH2A and ZmFtsH2B were identified, which were 4420 bp and 5187 bp in length respectively.Both genes have 5 exons and 4 introns and the main difference between the two sequences is that the first intron of ZmFtsH2B is much longer than that of ZmFtsH2A.DNA gel blot analysis showed that the two genes were single-copy in maize genome.RT-PCR analysis revealed that both maize FtsH genes were expressed in all the examined tissues including leaves,roots, stems,ears and tassels and the ZmFtsH2B transcript was higher than ZmFtsH2A in the common tissues.The responses of the two genes in leaves and roots of maize seedlings subjected to drought,cold,high salt,methyl jasmonate and abscisic acid treatments were compared using Real-time PCR approach and the results showed that ZmFtsH2B transcript was markedly up-regulated by osmotic and ABA stresses in leaves while ZmFtsH2A constitutively expressed both in leaves and roots under all tested stressful conditions.To investigate the physiological functions of two FtsH proteases,the full length cDNAs of ZmFtsH2A and ZmFtsH2B driven by 35S promoter were transformed into tobaccos.The presence and expression of the FtsH2 gene in To transgenic tobaccos were confirmed by PCR and RT-PCR approaches and drought tolerance analysis of transgenic plants were carried out.The results showed that drought tolerance of transgenic tobaccos overexpressing the two AtFtsH2-like genes were not improved compared to wild type controls,which indicated that maize FtsH2 might not be involved in plant drought tolerance.In our study,a forward cDNA library of seedling leaves treated with 18%PEG was also constructed by suppression subtractive hybridization,and a 764 bp partial cDNA of putative type-â…¡H⁺-PPase gene was obtained by screening the SSH-cDNA library.The 764 bp EST was used as the seed to search the NCBI EST database of Zea mays,and only 1511 bp long contig was obtained by in silico approach.This contig was not complete and lacked its 5' end.So its homologous gene of Oryza Sativa was found and used to search the Zea mays EST database as a new seed.Then an 898 bp contig containing its 5' end was obtained.So the possible 5' and 3' sequence of the gene was clear although the sequence between them was still unknown.Primers were designed according to the results of in silico cloning and full length for the cDNA was obtained by RT-PCR.In addition,its 3' UTR was cloned by 3'-RACE method.The isolated 2974bp full-length cDNA of maize type-â…¡H⁺-PPase contains a single 2400bp ORF encoding a putative protein of 799 amino acids.The predicted protein has five conserved domains and show 89%identity to Golgi apparatus resident type-â…¡H⁺-PPase(AVP2)from Arabidopsis thaliana,but has only 39%amino acid sequence identity to the maize vacuolar H⁺-PPase encoded by Vpp1 (GenBank accession no AJ715528).DNA gel blotting analysis showed that H⁺-PPase is a low-copy gene.Organ expression pattern analysis revealed that type-â…¡H⁺-PPase expressed highly in leaf and tassel,followed by in stem,root,and ear.The Real-time RT-PCR assays showed that the expression of the gene was up-regulated both in shoots and roots of maize seedlings under dehydration,cold and high salt stresses. Those results suggested that maize type-â…¡H⁺-PPase product may play an important role in abiotic stress tolerance of Zea mays.