Osmotic Regulation of Betaine Content in Leymus chinensis Under Saline-alkali Stress and Cloning and Expression of Betaine Aldehyde Dehydrogenase(BADH) Gene

The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different（from Ⅰ to Ⅵ） concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase（BADH： EC 1.2.1.8） activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leyrnus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH. The cDNA sequence of LcBADH was 1774bp including the open reading frame（ORF） of 1521bp（coding 506 amino acids）. The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragmcnt into pET30a（＋） and transformed into E. coli BL21（DE3）. The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl fl-D-thiogalactoside（IPTG）.

Molecular cloning, sequence analysis, and cadmium stress-rated expression changes of BTG1 in freshwater pearl mussel(Hyriopsis schlegelii)

The B cells translocation gene 1 （BTG1） is a member of the BTG/TOB family of anti-proliferative genes, which have recently emerged as important regulators of cell growth and differentiation among vertebrates. Here, for the first time we cloned the full-length eDNA sequence of Hyriopsis schlegelii （Hs-BTG1）, an economically important freshwater shellfish and potential indicator of environmental heavy metal pollution, for the first time. Using rapid amplification of eDNA ends （RACE） together with splicing the EST sequence from a haemocyte eDNA library, we found that Hs-BTG1 contains a 525 bp open reading frame （ORF） encoding a 174 amino-acid polypeptide, a 306 bp 5＇ untranslated region （5＇ UTR）, and a 571 bp 3＇ UTR with a Poly（A） tail as well as a transcription termination signal （AATAAA）. Homologne searching against GenBank revealed that Hs-BTG1 was closest to Crassostrea gigas BTG1, sharing 50.57% of protein identities. Hs-BTG1 also shares some typical features of the BTG/TOB family, possessing two well-conserved A and B boxes. Clustering analysis of Hs-BTG1 and other known BTGs showed that Hs-BTG1 was also closely related to BTG1 of C. gigas from the invertebrate BTG1 clade. Function prediction via homology modeling showed that both Hs-BTG1 and C. gigas BTG1 share a similar three-dimensional structure with Homo sapiens BTG1. Tissue-specific expression analysis of the Hs-BTG1 via real-time PCR showed that the transcripts were constitutively expressed, with the highest levels in the hepatopancreas and gills, and the lowest in both haemocyte and muscle tissue. Expression levels of Hs-BTG1 in hepatopancreas （2.03-fold）, mantle （2.07-fold）, kidney （2.2-fold） and haemocyte （2.5-fold） were enhanced by cadmium （Cd2＋） stress, suggesting that Hs-BTG 1 may have played a significant role in H, schlegelii adaptation to adverse environmental conditions.

Cloning and Expression Analysis of OsNADPH1 Gene from Rice in Drought Stress Response

An experiment was conducted to compare the mRNA expression difference in rice leaves and roots under drought stress and normal conditions us, ng Fluorescent Differential Display （FDD） method. One positive fragment was isolated by combination of the H. A. Yellow-PAGE （cont,~ined 0.1% H. A. Yellow） separation and macroarray screening methods. Compared to Arabidopsis thaliana NADPH oxidoreductase gene, it has 96% identity. The cDNA was 1423 bp, and contained a complete open reading frame of 1048 bp encoding a protein with 345 amino acid residues. Moreover, the gene expression level was higher under drought stress than that under normal conditions. The possible role of NADPH oxidoreductase gene under drought response was also discussed.

Cloning and Expression Analysis of a Mitogen-Activated Protein Kinase Gene OsMPK14 in Rice

Mitogen activated-protein kinases （MAPKs） are important components in signal transduction pathways responding to various biotic and abiotic stresses. An MAPK gene, OsMPK14 （GenBank Accession No. GQ265780） from rice （Oryza sativa L.）, was cloned by RT-PCR. The full-length cDNA of OsMPK14 consists of 1660 bp in size, containing an open reading frame of 1629 bp, which encodes a 542-amino-acid polypeptide and has a typical protein kinase domain and a phosphorylation activation motif TDY. Sequence alignment and analysis revealed that OsMPK14 was located on rice chromosome 5, and composed of nine exons and eight introns in the coding region. Semi-quantitative RT-PCR was performed to detect the expression patterns of OsMPK14 in rice shoots and roots under darkness, drought, high salinity, low temperature and abscisic acid treatments. The OsMPK14 mRNA was induced by abscisic acid, low temperature and high salinity, but weakly inhibited by drought. In addition, the expression of OsMPK14 was up-regulated in roots, but down-regulated in shoots by light. The results indicate that OsMPK14 could be implicated in diverse rice stimuli-responsive signaling cascades, and its expression might be regulated by multiple factors.

Molecular Cloning and Characterization of Three Novel Genes Related to Fatty Acid Degradation and Their Responses to Abiotic Stresses in Gossypium hirsutum L.

Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and GhMFP, respectively, were isolated from Gossypium hirsutum acc. TM-1. The phylogenetic analysis revealed that amino acid sequences of GhACXand GhMFP have the highest homology with those from Vitis vinifera, and Gh4CL has a closer genetic relationship with that from Camellia sinensis. Tissue- and organ-specific analysis showed that the three genes expressed widely in all the tested tissues, including ovules and fiber at different developing stages, with expressed preferentially in some organs. Among them, GhACX showed the most abundant transcripts in seeds at 25 d post anthesis （DPA）, however, GhMFP and Gh4CL have the strongest expression level in ovules on the day of anthesis. Based on real-time quantitative RT-PCR, the three genes were differentially regulated when induced under wounding, methyl jasmonate （MeJA）, cold, and abscisic acid （ABA） treatments. The characterization and expression pattern of three novel fatty acid degradation related genes will aid both to understand the roles of fatty acid degradation related genes as precursor in stress stimuli and to elucidate the physiological function in cotton oilseed metabolism.

Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile（Pt CAT） was cloned using RTPCR and rapid amplification of c DNA ends（RACE）. Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif（^（61）FNRERIPERVVHAKGAG^（77））, a proximal heme-ligand signature sequence（^（352）RLFSYSDP^（359））, and three catalytic amino acid residues（H^（72）, N^（145）, and Y^（356））. Pt CAT also contains two putative N-glycosylation sites（^（34）NKT^（36） and ^（437）NFT^（439）） and a peroxisome-targeting signal（^（511）AQL^（513））. Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA： first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

Molecular cloning, characterization, and antioxidant function of catalase in Lymantria dispar asiatic (Lepidoptera: Lymantriidae) under avermectin stress

The critical antioxidant catalase(CAT)breaks down hydrogen peroxide induced by environmental stresses.Here we cloned full length catalase cDNA from Lymantria dispar asiatic(LdCAT).Bioinformatic analyses showed that open reading frames of LdCAT contains 1524 bp,encoding 507 amino acids with molecular weight of 126.99 kDa,theoretical pI of 5.00,aliphatic index of 29.92,grand average of hydropathicity of 0.764,and instability index(II)of 46.56.Protein BLAST and multiple sequence alignment indicated that LdCAT had high identity with CAT from other insects,especially lepidopterans.In a phylogenetic analysis,LdCAT was most similar to CAT from Spodoptera litura and S.exigua.Quantitative realtime polymerase chain reaction showed that LdCAT transcripts in all instar larvae and the five tissues tested,verifying the ubiquity of LdCAT in L.disapr.Moreover,LdCAT of third instar larvae was significantly upregulated after they fed on avermectin at sublethal and LC10 doses.The highest relative transcript levels were found 2 h after an avermectin spray at LC90,and in the cuticula,rather than heads,fat bodies,malpighian tubes,and midguts after a spray avermectin at a sublethal concentration.The expression level of LdCAT under pesticide stresses here suggested that CAT is an important antioxidant enzyme of L.disapr defensing against pesticide stress and may be a good target for controlling this pest.

Isolation of a Plasmalemma Aquaporin Encoding Gene StPIP1 from Solanum tuberosum L. and Its Expression in Transgenic Tobacco

Aquaporin （AQP） belongs to a highly conserved group of membrane proteins considered as major intrinsic proteins, which facilitate water transport across biological membranes. The discovery of AQPs in plants has resulted in a paradigm shift in the understanding of plant-water relations, however, the potential relationship between the role of aquaporins in regulating plant water balance and drought tolerance still remains elusive. In this study, the gene encoding potato AQP cDNA, StPIP1 （GenBank accession no. DQ999080）, was cloned from the leaf of potato cultivar Gannongshu 2 by reverse transcription-PCR （RT-PCR）. Sequence alignment was made by BLASTn in GenBank, the phylogenetic analysis was conducted using PHYLIPWY, the 3D structure was predicted in Swiss-Model server. Subcellular localization of StPIP1 was performed by constructing CaMV35S-StPIP1-GFP and rd29A-StPIP1-GFP fusion proteins and transient expression in onion epidermis. To understand StPIP1 physiological functions in potato under various stress conditions, the StPIP1 gene in a reverse orientation was transformed into tobacco driven by the Cauliflower mosaic virus （CMV） 35S promoter. The expression levels of transgenic and wild-type plants were assessed under various abiotic stress conditions using semi-quantitative RT-PCR, and the morphological and physiological responses of transgenic plants to different stress conditions were investigated. The expression of StPIP1 mRNA decreased in transgenic plants under non-stress and stress conditions, however, the reduction was more severer under drought stress. In both non-stress and stress conditions, StPIP1 was expressed predominantly in root. The morphological and physiological investigation showed no significant differences in growth rate, germination rate, and root fresh weight （FW） between transgenic and wild-type plants when grown under favorable conditions. In contrast, under drought stress, the reduction in StPIPI expression leads to a delay in seed germination and seedling growth, accelerated seedling wilt, and leaf morphological abnormity. Under ＂enough＂ water conditions （i.e., water culture）, the aerial parts of anti-sense plants showed no differences. However, for the aerial parts to accumulate the same amount of biomass, transgenic plants needed about 3 times more abundant root system to transport water for plant growth than wild-type plants. Morphological investigation showed that the reduction in StPIP1 expression increased the root system in transgenic plants under drought stress. As a result, the increase of root mass might compensate the reduced cellular water permeability in order to ensure a sufficient water supply for the plant. Results demonstrated that StPIP1 plays an important role for water transportation in potato, especially under drought stress conditions. The reduced expression of StPIP1 decreases the cellular water transport and influences the expression of endogenous AQPs genes and thereby, has impacts on seed germination, seedling growth, and stress responses of potato to drought conditions.

Molecular cloning and characterization of a lipid transfer protein gene(PsLTP1) from Pinus sylvestris(L.)

Plant nonspecific lipid transfer proteins (nsLTPs) are widely distributed through plant kingdom and are characterized by the presence of a central hydrophobic cavity, suitable for binding various hydrophobic molecules. Despite extensive research on nsLTP in different plant species, mostly angiosperm, and the great diversity of physiological processes in which they seem to be involved, their exact functions still remain unclear. Also, very limited experimental data are available on nsLTP in gymnosperm. In this study, we report for the first time on the molecular cloning of nsLTP, from Pinus sylvestris L.(PsLTP1, GenBank accession JN980402.1) and the expression pattern of PsLTP1 during ontogenesis and in response to environmental stress conditions. Total RNA from roots of 7-day old pine seedlings was used to isolate the cDNA clone, corresponding to Scots pine lipid transfer protein. The open reading frame of PsLTP1 consists of 372 bp encoding a protein of 123 amino acids. Amino acid sequence alignment revealed that mature PsLTP1 shares high level of similarity with nsLTP from other conifers and with well-studied nsLTPs from angiosperms. The PsLTP1 contains a 27-amino-acid N-terminal signal sequence and presents all the features of a plant nsLTP. Amino acid comparison analysis and 3D structure prediction showed that PsLTP1 is a type 1 nsLTP. The results of the expression analysis of Scots pine PsLTP1 gene revealed that its transcripts accumulate in actively growing tissues. Furthermore, transcription of PsLTP1 was upregulated in response to cold and salt treatments, and downregulated during acidic, osmotic and water stresses.

青花菜多聚半乳糖醛酸酶基因BoPGX3的克隆及盐胁迫下的表达特征分析

多聚半乳糖醛酸酶(PG)可降解细胞壁,从而降低植物抵抗能力。为了解青花菜PG基因响应盐胁迫的分子机制,以青花菜自交系‘WN12-95B’为试验材料,提取青花菜的总RNA并合成cDNA,克隆PG基因,并检测其在盐胁迫下的表达特征。结果表明:青花菜BoPGX3基因序列全长1476 bp,推导编码491个氨基酸,相对分子量为53.44 kD,等电点为5.84。系统进化分析表明,青花菜BoPGX3与羽衣甘蓝、甘蓝、白菜、萝卜和花椰菜的PG基因亲缘关系较近。荧光定量PCR结果显示,BoPGX3基因受盐胁迫诱导表达,处理3 h后相对表达量达到初始表达量的2.9倍,处理12 h时相对表达量下降至初始表达量的1.4倍。综上,青花菜BoPGX3基因参与了盐胁迫的响应,并可能在此过程中发挥着重要的作用。

盐生草HgWRKY19和HgWRKY23基因的克隆及表达分析

【目的】WRKY转录因子在响应植物逆境胁迫过程中发挥重要的生物学功能。克隆盐生草(Halogeton glomeratus)HgWRKY19和HgWRKY23转录因子,分析其亚细胞定位及表达模式,为进一步探究其在盐胁迫响应中的分子机制奠定基础。【方法】基于盐生草全长转录组数据,采用RT-PCR法克隆得到HgWRKY19和HgWRKY23全长序列,通过烟草瞬时转化试验明确其亚细胞定位,并使用qRT-PCR方法分析其在不同盐胁迫时间下(0、6、24和48 h)盐生草叶片和根系中的相对表达量。【结果】成功克隆得到HgWRKY19和HgWRKY23基因,分别编码389和378个氨基酸;其编码蛋白均包含1个典型的WRKYGQK保守结构域和C2H2型(CX5C23HXH)的锌指结构,具碱性、不稳定性、亲水性、无信号肽和跨膜结构等特征;有65个磷酸化位点;二级结构预测均以无规则卷曲占比最大。系统进化分析发现HgWRKY19和HgWRKY23与藜麦、菠菜、甜菜头等藜科草本植物的WRKY蛋白同源性最高。亚细胞定位显示二者主要在细胞核和细胞膜中表达。qRT-PCR分析表明HgWRKY19和HgWRKY23主要在根系表达,具组织特异性,且表达量均在盐胁迫24 h达到峰值。【结论】HgWRKY19和HgWRKY23编码蛋白具典型的WRKY家族结构特征,属于Ⅱd亚类WRKY蛋白,进化高度保守,定位于细胞核和细胞膜上。HgWRKY19和HgWRKY23在根系中受盐胁迫诱导表达,且在不同胁迫时间下表达量差异显著(P<0.05),推测其在盐胁迫响应中发挥重要作用。

马铃薯PYL5基因对非生物胁迫的响应分析及其启动子的活性鉴定

【目的】脱落酸(ABA)作为一种“应激激素”在植物生长发育和响应干旱、盐等非生物胁迫过程中发挥重要作用,PYR/PYL/RCARs(以下简称“PYL”)作为ABA受体在多种植物中也被广泛研究。基于对马铃薯StPYL5基因生物信息学及表达模式分析,并通过对其启动子活性鉴定,为进一步揭示马铃薯StPYL5功能及抗逆育种提供依据。【方法】根据转录组数据克隆得到StPYL5基因,通过DNAMAN、MEGA等软件分析了StPYL5的分子特征;通过qPCR检测了StPYL5基因的组织特异性及其对非生物胁迫的响应;利用PlantCARE网站对StPYL5基因启动子进行了分析,并通过瞬时转化烟草对其活性进行了鉴定。【结果】StPYL5基因全长534 bp,共编码177个氨基酸,蛋白质分子量20.19 ku,理论等电点(pI)为5.97。系统进化分析显示,StPYL5与SpPYL9-like亲缘关系较近。组织特异性分析结果显示,青薯9号(QS9)中StPYL5在根和叶中表达量较高,其次分别是茎和花,在块茎中表达量较低。不同胁迫下StPYL5表达量分析表明,青薯9号(QS9)中StPYL5在干旱、低温、盐和ABA胁迫下的表达量先升高后降低,且StPYL5的表达受Me JA和SA的诱导。此外,笔者成功克隆得到2 000 bp的StPYL5基因启动子。在烟草中的瞬时转化后的组织化学染色结果表明,StPYL5基因启动子具有成功启动下游GUS报告基因表达的启动子活性。【结论】全面分析了StPYL5基因的分子特征及其在多种非生物胁迫下的表达谱,并成功克隆到具有活性的pStPYL5启动子,该结果为深入研究StPYL5基因的功能以及马铃薯抗逆育种提供依据。

小麦钙依赖蛋白激酶基因TaCDPK17的克隆及其抗逆功能初步分析

为探究钙依赖蛋白激酶(CDPK)在小麦生长和逆境应答中的作用,从普通小麦中克隆了TaCDPK17基因,并对其进行了序列结构、表达模式和抗逆功能初步分析。结果表明,TaCDPK17基因编码区长1701 bp,编码566个氨基酸,具有CDPK家族的典型结构特征,包括1个保守的丝氨酸/苏氨酸激酶结构域和4个EF手型结构域。对TaCDPK17与其他12种植物的CDPK17进行进化树分析表明,TaCDPK17与禾本科作物,特别是节节麦、大麦的CDPK17序列有较高同源性。TaCDPK17基因启动子区域富含与激素调控、光照响应,以及非生物应答胁迫相关的顺式调控元件,其中,脱落酸(ABA)响应元件(ABRE)和茉莉酸甲酯响应元件(CGTCA)数量较多。基于实时荧光定量PCR的表达分析结果显示,TaCDPK17受100μmol/L ABA、100μmol/L茉莉酸甲酯、20%PEG6000和250 mmol/L NaCl诱导后,表达量有不同程度升高。在2μmol/L ABA和100 mmol/L NaCl胁迫处理条件下,过表达TaCDPK17的拟南芥种子萌发率显著高于野生型对照。同时,过表达TaCDPK17缓解了ABA或渗透胁迫处理对幼苗根生长的抑制作用。在气孔关闭过程中,过表达TaCDPK17的转基因植物对ABA更敏感,与野生型植物相比,表现出更强的气孔关闭趋势。这些结果表明,TaCDPK17在小麦逆境胁迫和激素信号应答中发挥着重要作用。

烟草磷转运蛋白基因NtPHT2;1克隆和表达分析

【目的】植物对磷酸盐的吸收与利用主要依靠磷转运蛋白,其中PHT2家族编码的低亲和磷转运蛋白主要负责植物在正常供磷条件下磷酸盐的吸收、转运与再利用,为了探究低亲和磷转运蛋白基因NtPHT2;1在烟草转运磷酸盐中的作用和表达模式,通过NtPHT2;1培育烟草磷素高效利用新品种。【方法】以普通烟草K326的cDNA为模板,克隆得到NtPHT2;1,对该基因进行生物信息学分析和蛋白质的亚细胞定位,并通过荧光定量PCR技术对该基因在低磷等非生物胁迫下的基因表达模式进行分析。【结果】(1)NtPHT2;1基因全长1764 bp,编码587个氨基酸;(2)亚细胞定位结果显示,NtPHT2;1蛋白定位于叶绿体上;(3)同源性比对发现,NtPHT2;1蛋白与辣椒CaPHT2;1蛋白的同源性最高达到91.00%;(4)NtPHT2;1启动子含有参与调控植物衰老、逆境胁迫相关的顺式作用元件;(5)NtPHT2;1在叶片表达量最高,新叶中的表达量比老叶中高;在低磷诱导条件下,该基因表达量与正常条件相比差异不显著;(6)非生物胁迫下的表达模式分析显示,NtPHT2;1基因表达量在盐胁迫和干旱胁迫下显著降低。【结论】NtPHT2;1基因主要是负责烟株正常生长发育条件下磷酸盐的转运与利用。

蓖麻RcbZIP44基因克隆及表达特性

【目的】克隆蓖麻bZIP转录因子基因RcbZIP44,并进行表达特性分析,为探究bZIP转录因子在蓖麻生长发育及非生物胁迫下的生物学功能提供理论参考。【方法】从蓖麻品种通蓖5号中克隆bZIP转录因子基因家族成员RcbZIP44,对其序列进行生物信息学分析,借助烟草瞬时表达系统对RcbZIP44蛋白进行亚细胞定位,通过实时荧光定量PCR检测RcbZIP44基因在不同组织及非生物胁迫下的表达模式。【结果】克隆获得的RcbZIP44基因cDNA全长612 bp,包含1个456 bp的开放阅读框(ORF),编码151个氨基酸残基,无跨膜区域和信号肽结构,为不稳定的亲水性蛋白,存在21个潜在的磷酸化位点,二级结构以α-螺旋为主,定位于细胞核中。RcbZIP44基因启动子区域含有激素响应元件(ABRE、GARE-motif、TATC-box、TGACG-motif)、胁迫响应元件(LTR、ARE、MRE)等,推测RcbZIP44基因在非生物胁迫中发挥重要作用。RcbZIP44基因在茎中的相对表达量最高,显著高于根、子叶和真叶(P<0.05),说明其具有明显的组织表达特异性。在脱落酸(ABA)、盐、干旱和低温胁迫下,RcbZIP44基因在真叶和根中表现出不同的表达模式。RcbZIP44基因对低温胁迫敏感,处理6 h时的相对表达量最高;RcbZIP44基因在ABA和干旱胁迫下呈先增后减的表达模式,在处理12 h时的相对表达量最高;在盐胁迫中RcbZIP44基因表达较为平缓。【结论】RcbZIP44基因编码的蛋白含有bZIP家族特有的bZIP-plant-GBF1结构域,其主要在茎中发挥调控作用,且该基因受ABA、盐、干旱和低温等非生物胁迫诱导表达,推测RcbZIP44基因在蓖麻生长发育及响应非生物胁迫中发挥重要调控作用。

大蒜类钙调蛋白基因AsCML15和AsCML42的分离及其在渗透胁迫中的功能分析

类钙调素蛋白(CMLs)是植物中的Ca 2+感受器之一,参与植物的生长发育和适应环境变化的过程。为了解大蒜CML的序列特征及其对渗透胁迫的响应,从大蒜品种苍山四六瓣中克隆得到AsCML15和AsCML42基因,并对其在干旱以及盐胁迫条件下的表达模式进行了分析。结果表明,AsCML15和AsCML42基因开放阅读框长度分别为498,543 bp,编码165,180个氨基酸残基。AsCML15和AsCML42分别含有4,3个EF-hand结构域。AsCML42与拟南芥AtCML42和AtCML43在进化关系上更为接近,而AsCML15与拟南芥AtCML15、AtCML16亲缘关系较近。荧光定量PCR结果表明,AsCML15和AsCML42在鳞茎、叶片、根都有表达,且均能被200 mmol/L NaCl和15%PEG6000所诱导。AsCML15和AsCML42基因可能参与了大蒜抵御盐胁迫和干旱胁迫的过程,可进一步鉴定其生物学功能。

菊芋14-3-3基因家族的鉴定及其对非生物胁迫响应的分析

[目的]本文旨在鉴定菊芋14-3-3基因家族成员并分析它们对高温、低温、盐和干旱胁迫响应的表达模式,为研究14-3-3蛋白功能及菊芋育种提供依据。[方法]采用克隆和生物信息学研究基因性质,采用RNA-seq数据分析和RT-qPCR研究基因对非生物胁迫的响应模式。[结果]从菊芋中克隆到14-3-3基因家族的10个成员HtGRF1—HtGRF10,GenBank登录号为OP132618—OP132627。根据进化关系将其分为2个亚家族:HtGRF1—HtGRF7属于非ε组,HtGRF8—HtGRF10属于ε组,通常形成同源或异源二聚体。组织表达分析表明,HtGRF2/3/6/9在芽、根、茎、叶中的表达丰度较高,HtGRF3/5/9在块茎发育过程中前高后低。综合分析根和叶中HtGRF对非生物胁迫响应时发现,HtGRF6表达水平在高温、盐和干旱胁迫下下降;HtGRF2/3/7/8/9表达水平在盐胁迫下下降,而在干旱胁迫下上升;HtGRF1表达水平在低温胁迫下上升;HtGRF4/5表达水平在干旱胁迫下下降;而HtGRF10表达水平变化不显著。[结论]菊芋14-3-3蛋白是一个高度保守的多基因编码家族,在菊芋的生长发育和适应复杂环境中起着重要作用。

异源表达甜菜BvHSP18.2基因增强拟南芥对镉胁迫的耐受性研究

为进一步分析和验证甜菜BvHSP18.2基因(LOC104903994)受镉胁迫调控的功能,本研究用RTPCR技术克隆了甜菜BvHSP18.2基因,并通过植物表达载体构建以及拟南芥的遗传转化,测得异源表达目的基因的拟南芥植株在不同浓度镉胁迫下的表型及生理变化数据。研究结果表明,随着镉胁迫浓度的增加,异源表达BvHSP18.2基因的拟南芥无论在根长还是鲜重方面均具有优势:50μmol/L镉胁迫下平均根长相差最大,为1.1 cm,600μmol/L镉胁迫下平均鲜重相差最大,为0.015 g。同时,BvHSP18.2基因的过表达可有效提高拟南芥体内SOD和POD活性,300μmol/L镉胁迫下SOD活性最高,为657.7266953 U/g;600μmol/L镉胁迫下POD活性最高,为野生型拟南芥的1.91倍。BvHSP18.2基因的过表达增强了拟南芥在镉胁迫下的耐受性,推测该基因在植物对镉胁迫的适应机制中具有重要的作用。

楸子MpRZ 1基因的克隆及响应干旱胁迫表达分析

基于干旱胁迫下楸子(Malus prunifolia)转录组得到的4条RZs转录本,通过电子延伸和RT-PCR方法克隆得到1个新基因MpRZ 1,利用生物信息学方法对该基因编码蛋白的结构及功能特性进行预测,并与苹果(Malus domestica)RZ家族成员进行同源性比较,利用qRT-PCR技术验证干旱胁迫下MpRZ 1基因的表达模式。结果表明,MpRZ 1基因cDNA全长930 bp,开放读码框为843 bp,编码280个氨基酸残基,分子量为30.97 kDa,等电点为9.37,无N端信号肽,无跨膜结构阈,推测该蛋白可能定位于细胞核中,是一种不稳定的碱性亲水蛋白,含有38个潜在的磷酸化修饰位点,13个O-GlcNAc和5个N-Glyc潜在的糖基化修饰位点。该蛋白二级元件主要以无规卷曲和α-螺旋组成,两者占比达87.1%。MpRZ1蛋白N-端含有1个保守的RNA识别基序,由5个反向平行的β-折叠与2个α-螺旋排成β_(1)α_(1)β_(2)β_(3)α_(2)β_(4)β_(5)拓扑结构,C-端的甘氨酸富含区含有1个CCHC型锌指和一系列(GGX)n和(DRX)n重复序列。序列比对和聚类分析显示,MpRZ 1编码蛋白与拟南芥、水稻RZs蛋白有较高的序列相似性和亲缘关系,属于RZ基因家族成员。通过苹果基因组搜索得到9个推测的MdRZs基因,其编码蛋白均为碱性亲水蛋白,其中2个MdRZs编码蛋白(MdP0000088428、MdP0000272138)与MpRZ 1编码蛋白的序列相似性超过96%。楸子和平邑甜茶叶片中RZ基因的表达水平均在干旱胁迫9 d时达到峰值且与对照差异显著(P<0.05),表明MpRZ 1基因参与干旱胁迫的应答过程。

辣椒CcBBX20基因克隆和表达分析

锌指蛋白在植物生长发育和对非生物胁迫因子的响应中发挥着重要的调节作用。BBX蛋白属于锌指蛋白,CcBBX20基因是锌指蛋白家族的重要成员。为探讨CcBBX20在辣椒生长发育和响应非生物胁迫中的生物学功能,本研究利用基因克隆获得基因的编码序列;应用生物信息学分析方法预测该基因编码蛋白质的结构和功能;根据PepperHub数据库相关数据分析该基因在10种非生物胁迫处理下辣椒幼苗根和叶的表达量模式。结果表明:辣椒CcBBX20所编码的蛋白有209个氨基酸残基,相对分子质量为23.12 kD,分子式为C984H1551N285O329S15,理论等电点为5.78,是不稳定的亲水性蛋白;有2个典型的Bbox-SF结构域,没有信号肽和跨膜结构域,主要定位在细胞核中;其二级结构主要由无规则卷曲组成(占58.85%),α螺旋占比22.01%,β-转角占比3.83%。辣椒CcBBX20和马铃薯、番茄、茄子BBX20的亲缘关系最近。基因表达分析表明CcBBX20在辣椒幼苗根和叶受到不同胁迫后表达差异显著,在脱落酸、赤霉素、生长素、水杨酸、茉莉酸甲酯以及高温处理下表现为促进作用,其中高温42℃,处理1.5 h后表达量最高;在过氧化氢、甘露醇和盐处理下表现为抑制作用。因此辣椒CcBBX20基因作为BBX基因家族成员,在响应高温胁迫中发挥作用,可能会提高幼苗对高温的耐受力,可为辣椒耐高温育种目标提供候选基因。