黄瓜矮化突变体膨胀素Cs-EXPA2基因的克隆与表达分析

采用RT-PCR的方法克隆了黄瓜矮化突变体中膨胀素Cs-EXPA2基因,并对其苗期88 h内生长过程中mRNA水平表达量的差异进行了研究,探讨膨胀素基因对矮化的影响与作用.结果显示,Cs-EXPA2在黄瓜矮化突变体的下胚轴、根、子叶中均有不同程度的表达,利用Real-time PCR分析得出Cs-EXPA2的表达量随时空变化,Cs-EXPA2在下胚轴中的表达模式为"低-高-低",在根中的表达模式为"高-低-高".     

光质对矮化黄瓜膨胀素基因表达的影响

以矮生黄瓜"D0462"和蔓生黄瓜"129"为试材,研究不同光质对其膨胀素基因表达的影响。结果表明:不同光质对黄瓜下胚轴的伸长起到或抑制或促进的作用;通过Real-time PCR定量分析推测红光抑制膨胀素基因的表达;蓝光对膨胀素基因Cs-EXPA1,Cs-EXPA2的表达先抑制后促进;推断黄瓜"D0462"的矮化性状与膨胀素基因表达关系密切,其中Cs-EXPA1基因的影响尤为关键。     

黄瓜钙依赖性蛋白激酶基因CsCDPK5的克隆及响应淹水胁迫的表达分析

通过对黄瓜耐涝品系Zaoer-N和不耐涝品系Pepino淹水处理72 h后发现,Zaoer-N下胚轴平均可生成24.6条不定根,而Pepino平均仅能生成1.5条不定根,两者差异显著。从上述两个品种中克隆了钙依赖性蛋白激酶基因CsCDPK5的全长,两者序列完全一致,全长均为1 887 bp,编码629个氨基酸,预测其编码蛋白质分子量为59.17 k D,理论等电点为5.65。亚细胞定位预测表明CsCDPK5定位于细胞质。Zaoer-N和Pepino启动子相似度为99%,有14个碱基位点的差异,两者均含有与激素信号及低氧胁迫响应相关的顺式作用元件,而诱导子激活元件AT-rich sequence和功能未知元件F-box为Zaoer-N所特有的启动元件。qRT-PCR结果表明:(1)CsCDPK5在黄瓜下胚轴中的表达量最高;(2)淹水条件下,Zaoer-N和Pepino下胚轴中CsCDPK5的相对表达量均呈现先上升后下略有降,之后又上升的趋势,且Zaoer-N中的表达量大于Pepino;(3)根和叶中CsCDPK5的相对表达量无明显规律性;(4)在使用乙烯竞争性抑制剂1-MCP预处理后,淹水条件下Zaoer-N下胚轴不定根形成被抑制,CsCDPK5的表达与非淹水对照亦无明显差异。综上所述,黄瓜CsCDPK5为淹水胁迫响应基因,可能参与黄瓜淹水后下胚轴不定根的形成过程。     

黄瓜3-磷酸甘油醛脱氢酶基因CsGAPDH的克隆及其涝胁迫响应分析

以黄瓜耐涝品系‘早二N’根组织总RNA为模板,运用RT-PCR结合RACE技术,获得了黄瓜3-磷酸甘油醛脱氢酶基因（CsGAPDH）的cDNA全长序列,基因编码区共1011bp,编码336个氨基酸（GenBank登录号HQ156465）。系统进化分析表明：CsGAPDH基因与胡萝卜、葡萄、大豆等双子叶植物同源基因的核苷酸序列相似性均在88%以上,与单子叶植物水稻和玉米同源基因的相似性达86.8%和86%。qRT-PCR表达分析结果表明：CsGAPDH基因快速响应涝胁迫,在涝胁迫2h,该基因在根中表达量达到对照水平的12倍,在涝胁迫12h达到表达高峰,随后表达量呈现逐渐下降趋势;CsGAPDH基因在‘早二N’的根、茎、叶中均有表达,根和叶中原始表达量高于茎,在涝胁迫4h后均诱导表达,根中诱导表达量上调幅度最大,为初始表达量的4倍。CsGAPDH属于黄瓜涝胁迫响应基因,在缓解涝胁迫伤害过程中可能具有重要的调控作用。     

大岩桐SsPIN2基因的克隆及组织表达分析

以大岩桐为试材,采用RT-PCR方法对大岩桐SsPIN2基因进行了克隆,获得898bp的基因序列,经BLAST比对,采用邻接法构建进化树;并利用半定量RT-PCR方法,研究了SsPIN2基因在大岩桐叶、茎尖、根、茎、花瓣和花萼组织的表达情况。结果表明:大岩桐SsPIN2基因与黄瓜CsPIN2、豌豆PsPIN2基因等亲缘关系较近;该基因在上述6种组织中均有表达组织,其中在茎尖、根、茎和花萼的表达量较高,在叶和花瓣中的表达量较低。     

黄瓜3–磷酸甘油醛脱氢酶基因CsGAPDH的克隆及其涝胁迫响应分析

 以黄瓜耐涝品系‘早二N’根组织总RNA为模板,运用RT-PCR结合RACE技术,获得了黄瓜3–磷酸甘油醛脱氢酶基因（CsGAPDH）的cDNA全长序列,基因编码区共1 011 bp,编码336个氨基酸（GenBank登录号HQ156465）。系统进化分析表明：CsGAPDH基因与胡萝卜、葡萄、大豆等双子叶植物同源基因的核苷酸序列相似性均在88%以上,与单子叶植物水稻和玉米同源基因的相似性达86.8%和86%。qRT-PCR表达分析结果表明：CsGAPDH基因快速响应涝胁迫,在涝胁迫2 h,该基因在根中表达量达到对照水平的12倍,在涝胁迫12 h达到表达高峰,随后表达量呈现逐渐下降趋势;CsGAPDH基因在‘早二N’的根、茎、叶中均有表达,根和叶中原始表达量高于茎,在涝胁迫4 h后均诱导表达,根中诱导表达量上调幅度最大,为初始表达量的4倍。CsGAPDH属于黄瓜涝胁迫响应基因,在缓解涝胁迫伤害过程中可能具有重要的调控作用。     

梨极矮化突变体与苹果梨GA20ox基因的克隆及植物表达载体的构建

以梨极矮化突变体和苹果梨为试材,应用RT-PCR技术对GA20ox基因进行克隆及序列分析。结果表明:克隆得到梨极矮化突变体和苹果梨GA20-氧化酶基因,依次命名为PuGA20ox和PbGA20ox,其全长均为1 179bp,分别包含1个完整编码392个氨基酸的开放阅读框(ORF);对其进行序列分析,并成功构建了植物表达载体,为进一步研究梨矮化突变体和GA20-氧化酶基因功能奠定了基础。     

EMS诱变的‘里格尔87-5’番茄M_2群体突变体表型及其抗坏血酸研究

构建番茄突变体库对于丰富遗传变异、发掘基因资源具有重要意义。利用1%的甲基磺酸乙酯(EMS)处理‘里格尔87-5’加工番茄种子12 h,对M_2代材料进行农艺学性状与生物学性状鉴定,对部分M_3代材料进行遗传稳定性验证。在M_2代筛出典型突变体,包括早衰、矮化、圆果、低抗坏血酸、黑斑、绿茎、簇生等突变单株。对240个M_2代家系中共1 023个单株全生育期田间表型进行观察鉴定和果实品质测定,共发现90个变异单株,其中13个突变单株同时出现两个或两个以上变异性状,共50个变异性状,总的单株变异频率(突变株/总株数)为8.80%。叶、茎、花、果实和植株发生的变异株数分别为27、7、17、56和21,性状变异频率分别为2.64%、0.68%、1.66%、5.47%和2.05%。其中矮化突变体分子鉴定表明,curl-3基因外显子中C变成T,氨基酸由L变成F,喷施油菜素内酯不能恢复其正常生长,表明curl-3突变导致油菜素内酯信号传导受阻。curl-3突变体叶片与果实中抗坏血酸含量下降,并伴随着叶片中氧化态与还原态抗坏血酸比值增加,这可能与抗坏血酸氧化酶基因AO表达量和AO酶活性增强有关。     

外源硅对不同砧木嫁接黄瓜果面蜡粉和硅吸收及相关基因表达的影响

为探明砧木影响嫁接黄瓜果面蜡粉形成的机制以及与外源硅的关系,以‘山农5号’黄瓜为接穗,‘云南黑籽南瓜’(去蜡粉能力弱)和‘黄诚根2号’南瓜(去蜡粉能力强)为砧木进行嫁接,采用水培法,研究营养液中添加硅与否对黄瓜硅吸收、硅转运蛋白相关基因表达和果实表面蜡粉形成的影响。结果表明:加硅显著提高了‘云南黑籽南瓜’嫁接黄瓜和自根黄瓜果面蜡粉量及各器官硅含量,但加硅后‘黄诚根2号’嫁接黄瓜的果面蜡粉量和各器官硅含量均较低,与不加硅条件下的嫁接和自根黄瓜相近;加硅处理后‘云南黑籽南瓜’嫁接黄瓜苗根系中硅转运蛋白基因Cm Lsi1、Cm Lsi2-1、Cm Lsi2-2和Cm Lsi3的表达量均高于‘黄诚根2号’嫁接黄瓜苗;随着硅处理时间延长,‘云南黑籽南瓜’嫁接黄瓜苗根系中硅转运蛋白基因表达下调,而‘黄诚根2号’嫁接黄瓜苗根系中硅转运蛋白基因的表达同样下调。     

Molecular cloning and expression analysis of the gene encoding sedoheptulose-1, 7-bisphosphatase from Cucumis sativus

Sedoheptulose-1, 7-bisphosphatase (SBPase; EC 3.1.3.37) is a key enzyme in the photosynthetic carbon reduction cycle (Calvin cycle). In the present study, a full-length cDNA encoding SBPase (designated as CsSBP, GenBank accession no. FJ911553) was isolated from cucumber (Cucumis sativus L. cv. ‘Jinyou 3’) grown in solar-greenhouse by RT-PCR and RACE. The cDNA contained 1452 nucleotides with a complete open reading frame (ORF) of 1167 nucleotides, which was deduced for encoding a peptide of 388 amino acids whose molecular mass was inferred to be 42 kDa with its isoelectric point at 6.17. Sequence comparison analysis showed that the deduced amino acid of SBPase from cucumber leaves had high homology to that from other species. Expression analysis by real-time PCR and Western blot revealed that CsSBP mRNA and protein was most abundant in leaves, detectable in stems and fruits, but invisible in roots. A similar difference was found in SBPase activity among leaves, stems, fruits and roots. CsSBP expression at the mRNA and protein level was the highest in mid-position leaves (4th–8th apical expanded leaves) of plants, medium in base position leaves (12th), and the lowest in upper position leaves (1st). However, SBPase activity decreased successively as leaf position declined. The net photosynthetic rate (Pn) of mid-position leaves was also found to be the highest, but that of base-position leaves was lower than upper-position leaves. The diurnal variations of gene expression and SBPase activity in optimal functional leaf (6th leaf) was a single-peak pattern curve, which was similar to that of Pn, and the peak appeared at 12:00. These data indicate that CsSBP expresses only in green tissues of cucumber plants and there are close positive correlations between SBPase activity and gene expression at the mRNA and protein levels, and between SBPase activity and Pn in leaves of cucumber plants.     

短期亚适宜温光对黄瓜氮吸收运转相关酶活性和基因表达的影响

 以‘中农106’黄瓜为试材,对其进行不同温度和光照强度[正常温光25/15 ℃,光强（700 ±30）μmol · m^-2 · s^-1;亚适宜温光18/12 ℃,光强（200 ± 20）μmol · m^-2 · s^-1;低温弱光12/8 ℃,光强75 ~100 μmol · m^-2 · s^-1]处理,研究亚适宜温光条件对结果期黄瓜生长、生理特性和氮（N）吸收的影响。结果表明,与正常温光处理相比,亚适宜温光和低温弱光处理下黄瓜株高、叶面积、果实大小与产量均明显下降,根系活力、叶绿素含量、果实品质和谷氨酰胺合成酶（GS）、谷氨酸合成酶（GOGAT）、谷氨酸脱氢酶（GDH）活性也随处理温度和光照强度降低而降低。亚适宜温光条件下,结果期黄瓜根系、叶片、果实中N 含量均有所下降,亚适宜温光和低温弱光处理降低了结果期黄瓜N 运转蛋白基因的表达,且温度和光照强度越低,黄瓜根系、叶片和果实中N 运转蛋白基因表达量越低。说明亚适宜温光可能通过降低N 运转蛋白基因的表达,抑制了N 的吸收,降低N 含量,进而抑制了黄瓜生长。     

组织培养条件下番茄4 个品种对NaCl 胁迫耐受性的研究

以番茄品种白果强丰、红牛心、小红仙桃、红圣女的下胚轴为外植体,在培养基中分别添加0、50、100、150 mmol·L^-1 和200 mmol·L^-1 NaCl,研究在不同浓度NaCl 胁迫下番茄愈伤组织诱导率与不定芽诱导率的变化,评价4 个品种番茄在组织培养条件下对NaCl 胁迫的耐受性。同时探讨氯气熏蒸法用于番茄种子消毒时,熏蒸时间对番茄种子萌发与生长的影响。结果表明：氯气熏蒸番茄种子7 h 的消毒效果最好。4 个品种番茄的下胚轴组织培养能力和对NaCl 的耐受性均表现不同,白果强丰和红圣女的愈伤组织诱导率和不定芽诱导率较高;当以NaCl 作为抗性筛选剂对转化后代进行筛选时,适宜筛选浓度白果强丰为100 mmol·L ^-1、红圣女为50 mmol·L ^-1。     

null

 ‘台绿1 号’是分别以日本青花菜品种‘绿雄90’和‘Br60’为原始材料经连续多代自交选 育的自交不亲和系‘B19-10-1-2-1’和‘Br60-2-2-1-2’为母本和父本杂交育成的青花菜新品种。生长势较 强，植株半直立，叶片长椭圆形，叶色深绿，蜡粉中等。花球紧实，高圆形且圆整，蕾粒中细，单球质 量0.76 kg，产量约23 000 kg · hm^-2。中晚熟，从定植到采收约95 d，保鲜和速冻加工兼用。适合长江流域秋、冬季栽培。     

两个柱花草品种根系构型差异及其对间作柑橘砧木实生苗磷营养的竞争

 根系构型与植物的养分吸收密切相关,可能影响植物不同个体间的养分竞争。比较了‘格 拉姆’和‘184 号’柱花草（Stylosanthes Sw.）的根系构型差异,并探讨了两者在间作条件下对柑橘砧木 红藜檬（Citrus limonia）实生苗的生长和养分吸收的影响。结果表明,‘格拉姆’和‘184 号’在单作条 件下生物量、总根长、主根长和根尖数相似,但是‘184 号’的基根角度、根系表面积、平均根直径均小 于‘格拉姆’;在与柑橘实生苗间作条件下,‘184 号’有41.4%的根系进入柑橘根区,而‘格拉姆’仅有 0.8%的根系进入柑橘根区,因此与‘184 号’间作的柑橘砧木实生苗的生物量和对磷养分的吸收均显著低 于与‘格拉姆’间作的柑橘砧木实生苗。本研究表明,与根系垂直生长型的‘格拉姆’相比,根系水平 生长型的柱花草‘184 号’不宜用于果园生草栽培,根系构型应该成为草种筛选的重要指标。     

成熟黄瓜果皮红色性状的遗传分析及其基因定位

 以黄瓜（Cucumis sativus L.）成熟瓜红色果皮自交系‘NCG127’（P₁）和成熟瓜黄色果皮自交系‘9930’（P₂）为试验材料构建F₂遗传群体,对成熟瓜红色果皮R基因进行遗传分析和基因定位研究。结果表明,黄瓜成熟瓜红色果皮性状由显性单基因控制,红色对黄色为显性。以256株F₂分离群体为试材,应用群体分离分析（BSA）法筛选得到与R基因连锁的20个多态性SSR标记,构建了R基因的分子标记连锁图谱,将R基因定位到黄瓜4号染色体上,物理距离为213.4 kb的区段内,两侧翼标记为UW019319和UW019203,与R遗传距离分别为0.8 cM和0.7 cM。生物信息学分析表明,该区段存在30个预测候选基因。     

Effect of the expression level of an AGAMOUS-like gene on the petaloidy of stamens in the double-flowered lily, ‘Elodie’

Double-flowered lilies, in which stamens are converted to petaloid organs, are valuable for horticulturists. ‘Elodie’ is a double-flowered lily cultivar in which stamens are homeotically converted into petaloid organs in whorl 3. The ‘Elodie’ cultivar shows individual variation in stamen structure and it was therefore classified into the following three types according to the strength of petaloidy of the stamens: weak (type-I), intermediate (type-II), and strong (type-III) phenotypes. The AGAMOUS (AG) gene is a class C floral identity gene in Arabidopsis thaliana that is involved in the formation of stamens and carpels. An AG-like gene was isolated from ‘Elodie’ (LelAG1) and its expression was compared between flower types. The LelAG1 gene was expressed in whorls 3 and 4, but not in whorls 1 and 2 in all flower types. In type-I flowers, LelAG1 was expressed strongly in whorls 3 and 4, while its expression was significantly decreased in whorl 3 in type-III flowers. In type-II intermediate phenotype flowers, the expression level of LelAG1 in whorl 3 was reduced by 60%. These results suggest that the expression level of AG-like genes is correlated with the degree of petaloidy of the stamens.     

Effect of NaCl salinity in the genotypic variation of cowpea (Vigna unguiculata) during early vegetative growth

Twenty-five genotypes of cowpea (Vigna unguiculata L. Walp.) were tested for salt-tolerance at the vegetative growth stage in pot in the greenhouse experiments at salinity levels of 0, 85, and 170 mM NaCl. Plant survival was the main criterion for classifying genotypes. Other criteria included the ion concentration (Na⁺ and Cl⁻) in root and shoot and biomass accumulation. Four local accessions (‘Paceño’, ‘Tardón’, ‘Sonorense’, and ‘Cuarenteño’), three accessions from California (‘CB46’, ‘CB27’, and ‘CB3’), and one accession from the International Institute of Tropical Agriculture (IITA) (‘IT82D-889’) survived at concentrations of both 85 and 170 mM NaCl and were classified as salt-tolerant, while ‘IT96D-666’, ‘IT89KD-288’, and ‘IT93K-734’ from IITA were classified as salt-sensitive. One local accession (‘Sesenteño’), three accessions from IITA (‘PEPH-V Wes-85’, ‘IT86D-719’, and ‘IT95K-1090-12’), and one accession from California (‘CB5’) were classified as moderately salt-tolerant. Eight accessions from IITA (‘IT96D-733’, ‘IT90K-277-2’, ‘IT91K-93-10’, ‘IT91K-118-20’, ‘IT90K-284-2’, ‘IT95K-1088-4’, ‘IT89KD-391’, and ‘IT94K-437-1’) and one from California (‘CB88’) were classified as moderately salt-sensitive. Biomass was affected by both 85 and 170 mM NaCl in all groups of genotypes, however, salt-tolerant and moderately salt-tolerant genotypes showed higher biomass than genotypes classified as moderately salt-sensitive and salt-sensitive. In all genotypes Cl⁻ concentration was higher in shoots than roots and increased as salinity increased. Similarly Na⁺ concentration increased with increasing salinity. However, in salt-tolerant and moderately salt-tolerant genotypes, Na⁺ concentration was more in roots than shoots, while in moderately salt-sensitive and salt-sensitive genotypes, Na⁺ was higher in shoots than roots.