摘要
玉米(Zea mays L.)不仅是重要的粮食和饲料作物,也是重要的食品工业原料和能源植物。在我国,玉米产量仅次于水稻,在国民经济中占有非常重要的地位。干旱胁迫是自然界中主要的非生物胁迫之一,严重影响植物生长及作物生产。我国有60%的玉米种植面积受到干旱胁迫,每年因旱灾而减产20%～30%,直接影响国民经济发展及灾区人民生活。在玉米的生育期中,玉米苗期相对而言较为耐旱,适度的苗期干旱处理还可促进根系发育,增加玉米产量。而开花期前后受到干旱胁迫会严重影响玉米的产量。抽雄开花期是玉米对干旱最为敏感的时期之一,搞清楚在这一时期干旱对玉米的伤害及玉米对干旱的适应和调节机制,对于培育抗旱玉米品种具有重要意义,也是国内外学者关注的重要课题之一。
本研究以抽雄开花期的玉米为材料,利用玉米全基因组芯片检测了叶片在玉米植株受到干旱胁迫1天和7天时的基因表达谱;利用抑制性消减杂交技术构建了1天和7天干旱胁迫下抽雄开花期玉米叶片的两个消减文库并对部分阳性克隆进行了测序分析。本研究还克隆了玉米基因ZmFtsH2A、ZmFtsH2B和Ⅱ型H~+-PPase全长,并对它们进行了初步分析。
主要实验结果如下:
利用玉米全基因组芯片(microarray)检测了抽雄开花期玉米在1天和7天干旱胁迫下叶片中基因表达谱的变化情况。该芯片包含了57452个玉米寡核苷酸探针,代表了3万多个基因。以处理和对照样品的信号值的比值不低于2倍的探针为有意义的差异表达基因,发现在1天和7天两种干旱胁迫处理的玉米叶片中分别有195个和1008个基因呈现差异表达。在1天胁迫下的叶片中有102个基因表现上调,93个基因表现下调,上调的基因数占差异表达基因总数的52%;而在7天胁迫的叶片中有332个基因表现上调,676个基因表现下调,上调基因数只占33%。在两种处理下的叶片中则共有1144个基因呈现差异表达,其中有上调基因412个,下调基因732个,而在两种胁迫的叶片中均表现上调和均表现下调的基因分别只有22个和37个。通过对这些差异表达基因的生物信息学分析,发现在干旱胁迫1天表达显著上调的基因中,约有1/3属于信号传导类基因,参与细胞不同信号转导途径,表明信号传导相关基因在玉米对干旱胁迫的早期反应中起重要作用。在7天干旱胁迫下,大量的代谢相关基因差异表达,特别是渗透保护因子代谢相关基因的表达变化,如脯氨酸合成途径中的P5CS和P5CR基因表达上调而其降解途径的关键酶基因ProDH表达下调,海藻糖合成途径中的TPS和TPP表达下调而其降解途径的酶基因Trehalase表达上调,而参与棉子糖合成代谢的多个重要酶基因均受胁迫诱导上调表达。该工作表明,干旱对抽雄开花期玉米叶片基因表达的影响是复杂的,涉及到多条代谢途径,较长时间的干旱处理引起大量代谢相关基因差异表达,并且以下调表达为主;脯氨酸积累和糖代谢变化在玉米叶片对干旱胁迫的应答中起重要作用。本实验所得芯片杂交数据已提交至NCBI的GEO数据库(Gene Expression Omnibus database),获得的注册号为:GSE10596。
分别以1天干旱和7天干旱处理的抽雄开花期玉米叶片cDNA为tester,正常生长的玉米叶片cDNA为driver,使用抑制性消减杂交技术构建了两个干旱胁迫下玉米叶片的消减文库。两个文库的重组率均高于95%,插入片段集中在300—600bp之间。对两个文库部分克隆进行测序发现,文库中含有脱水素、蔗糖合成酶、甜菜碱醛脱氢酶、DRE结合因子等大量的抗旱相关基因,说明两个干旱胁迫下抽雄开花期玉米抑制性消减文库已经构建成功,且具有重要意义。
在抽雄开花期玉米叶片消减文库阳性克隆测序结果中,得到一条长为192bp的EST,使用同源性分析软件BLASTN检索NCBI的GenBank数据库发现该EST是玉米FtsH蛋白酶基因的部分cDNA序列。以该EST序列为种子探针,检索玉米EST数据库,将得到的匹配序列进行拼接,得到两条contig(重叠群)序列,分别长为2510bp和2430bp,两条序列在核苷酸水平上的相似性为95%,均含有2034bp的完整的开放读码框(open reading frame,ORF),是玉米的AtFtsH2-like(类拟南芥FtsH2)基因,被分别命名为ZmFtsH2A和ZmFtsH2B。根据电子克隆结果,分别在ZmFtsH2A和ZmFtsH2B的ORF两侧设计基因特异性引物,以玉米cDNA为模板,PCR扩增得到含有ORF的cDNA片段,测序结果证实了电子克隆的正确性。将ZmFtsH2A和ZmFtsH2B的cDNA序列提交至GeneBank数据库,获得的登录号为EU257690与EU257691。ZmFtsH2A的cDNA序列长2510bp,包含217bp的5′非翻译区(UTR)和241bp长的3′非翻译区(UTR)以及一个2034bp的开放读码框(ORF);与其相类似,ZmFtsH2B的cDNA序列长2430bp,包含222bp的5'UTR和56bp长的3'UTR及2034bp长的ORF。两个基因均编码677个氨基酸,其蛋白序列相似性高达97%且均存在AAA结构域和Zn~(2+)结合结构域等已知的金属蛋白酶FtsH家族的特征结构域。这两个基因的全长基因组序列分别长为4420bp(ZmFtsH2A)和5187bp(ZmFtsH2B),均由5个外显子和4个内含子组成,所有内含子的边界都具有真核生物典型的内含子切除位点GT/AG序列,编码区所在的外显子部分长度完全一样,而两个基因最大的区别在于ZmFtsH2B的第1内含子(1785bp)远长于ZmFtsH2A的第1内含子(913bp)。Southern杂交结果表明两个基因在玉米基因组中均是单拷贝的。表达谱分析表明两个基因在玉米的根、茎、叶、和雌雄穗中都有表达,但ZmFtsH2B在各组织中的表达量均明显高于ZmFtsH2A。在PEG、盐、冷、ABA和MeJA处理下,ZmFtsH2A基因在玉米根和叶片组织中的表达变化水平均没有达到2倍,而ZmFtsH2B基因仅在玉米叶片中受渗透胁迫和外源ABA的诱导上调表达,在其它情况下呈现组成型表达。为了分析ZmFtsH2A、ZmFtsH2B基因的生理功能,通过转基因技术分别获得了分别转ZmFtsH2A和ZmFtsH2B基因的烟草植株,并对转基因植株进行了耐旱性分析。初步结果表明:两个AtFtsH2-like基因在烟草中表达并没有提高烟草植株的耐旱性。
本论文还构建了18%PEG处理下苗期玉米叶片的消减文库,对部分文库进行测序分析时得到了玉米Ⅱ型H~+-PPase基因的长为764bp的EST序列。以该EST为探针,检索玉米的EST数据库,经几轮电子延伸后得到仅是该基因3′端序列的1511bp长的contig。接下来从水稻的核酸数据库中找出这条contig在水稻中的同源基因,并以该基因的5′端序列为探针,再次检索玉米的EST数据库,将匹配的玉米ESTs进行拼接后得到另一条长为898bp的contig,该contig含有该基因的5′端序列信息。这样,我们电子克隆出玉米Ⅱ型H~+-PPase基因的两端序列(中间序列未知),并依此设计基因特异性引物通过RT-PCR技术获得了该基因完整ORF的cDNA片段,并进一步利用3′RACE方法克隆了该基因的3′非翻译区序列。玉米Ⅱ型H~+-PPase基因全长2974bp,包含一个长2400bp的通读框,编码含779个氨基酸残基的蛋白质序列。该氨基酸序列同样具有H~+-PPase蛋白的5个保守结构域,与拟南芥的Ⅱ型H~+-PPase(AVP2)在氨基酸水平上具有89%的序列相似性,而与玉米Ⅰ型H~+-PPase相似性却只有39%。Southern杂交结果表明Ⅱ型H~+-PPase在玉米基因组中是低拷贝的。表达谱分析表明该基因在根、茎、叶、和雌雄穗中都有表达,其中在叶片和雄穗中表达强度比较高。Real-time PCR实验结果表明该基因的转录水平受缺水、高盐和冷胁迫等非生物胁迫诱导上调,显示该基因可能参与植物对多种非生物胁迫的耐受反应,克隆的玉米Ⅱ型H~+-PPase基因可能有较好的应用前景。
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 typeⅡH~+-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 Zn~(2+)-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-ⅡH~+-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-ⅡH~+-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-ⅡH~+-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-ⅡH~+-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-ⅡH~+-PPase product may play an important role in abiotic stress tolerance of Zea mays.
引文
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