马铃薯卷叶病毒外壳蛋白基因克隆转化 及其转基因后代的表达

根据GenBank中的马铃薯卷叶病外壳蛋白基因(PLRV-CP)全序列，设计一对特异性引物，以马铃薯卷叶病毒RNA为模板，克隆了马铃薯卷叶病外壳蛋白基因，在pBI121的基础上构建了植物表达载体。利用PLO(Poly-L-Ornithine)将PLRV-CP基因导入到马铃薯加工型品种大西洋的原生质体中，获得了转基因后代。在5株转化后代中扩增出长度为610bp的目标DNA片断，说明导入的外源PLRV-CP基因已经整合到马铃薯基因组中。Southern blot分析结果进一步证明了PCR结果的正确性。RT-PCR结果表明，在3株转化后代叶片中具有阳性表达。马铃薯卷叶病毒接种实验结果表明，转基因植株比对照有明显的马铃薯卷叶病抗性     

黄色镰刀菌侵染马铃薯块茎的组织细胞学观察

马铃薯干腐病会导致马铃薯在窖藏过程中发生腐烂,影响薯块的商品价值和食用价值。黄色镰刀菌(Fusarium culmorum)为黑龙江省马铃薯干腐病的主要致病菌。为观察镰刀菌对马铃薯块茎的侵染过程,对不同抗性的马铃薯品种(克新1号和大西洋)块茎在黄色镰刀菌侵染过程中的组织细胞学进行观察,结果表明,侵染2d时,马铃薯薯块内部出现了菌丝和孢子的积累;侵染16d时,薯块严重腐烂,其中克新1号块茎的病斑面积约占44.44%,大西洋的病斑面积约占89.34%。在显微结构观察中发现,侵染3d时,黄色镰刀菌已经在马铃薯块茎中大量积累,在侵染过程中薯块细胞的细胞壁呈现加厚和破损的现象,抗性马铃薯品种的受害病症出现相对较晚。克新1号相对大西洋表现出较强的抗病性。     

芒果细菌性角斑病菌在侵染芒果叶片过程中内参基因筛选

为筛选芒果细菌性角斑病菌在侵染芒果叶片过程中稳定表达的内参基因,采用实时荧光定量聚合酶链式扩增反应技术检测和分析病原细菌的16S rRNA、gyrB、GAPDH、dan K和rpo D这5个看家基因的表达量。结果表明,经过PCR扩增效率筛选,5个基因均扩增出单一条带,且条带大小与预期产物相符,特异性较好,符合基本稳定性要求。利用ge Norm软件分析发现,GAPDH和rpo D稳定性最高,M值均为0.122。其余3个基因的表达稳定性依次为gyr B (M=0.17)dan K (M=0.325)16S rRNA (M=0.542)。在5个候选内参基因中,GAPDH和gyr B为病原细菌侵染芒果叶片下表达最稳定的基因,且不需要引入第三个基因(V_(2/3)=0.063)。NormFinder软件分析5个候选内参基因稳定性数值顺序依次为:gyr B (0.092)dan K (0.169)GAPDH(0.188)rpo D (0.235)16S rRNA (0.581)。Bestkeeper程序分析5个候选内参基因的标准差(SD)值都在0.86～0.79之间。综合以上结果发现GAPDH和gyr B基因的表达稳定性最好,能够作为病原细菌在侵染芒果叶片时的内参基因,更准确地校正定量结果。     

小麦原生质体的电激介导基因转移

从小麦品种“Ｂｏｄａｌｉｎ”胚性悬浮细胞分离出原生质体，通过电激将质粒ＰＢＣ１ＤＮＡ（携带β－葡萄糖苷酸酶（ＧＵＳ）标记基因和潮霉素抗性基因ｈｐｈ）导入原生质体。采用ＢＴＸ电激系统和ＡＳＰ电激缓冲液，最佳电激条件为３００Ｖ（７５０Ｖ／ｃｍ）和５０ｍｓ（约１０００μＦ），转化的原生质体内ＧＵＳ的活性最高；质粒ＤＮＡ的有效使用浓度为２５μｇ／ｍｌ。电激处理后，原生质体培养２～３天，ＧＵＳ基因表达最强，宜于检测其瞬时表达；牛胸腺ＤＮＡ可协助提高ＧＵＳ基因的导入效果。质粒ＰＢＣ１ＤＮＡ处理的原生质体培养于添加潮霉素的ＫＭＰ培养基。经４个月抗性筛选，选择获得１５个潮霉素抗性克隆     

有机磷农药降解菌—阴沟肠杆菌的生物学特性

【研究目的】对有机磷农药降解菌阴沟肠杆菌的生物学特性进行研究,为研究其降解特性机理及应用提供理论依据。【方法】测定阴沟肠杆菌生长曲线、生理生化特征,筛选出最适生长培养基,测定不同温度、pH值、摇床振数、装液量下的生长状况,确定最适生长条件,测定对乐果、甲胺磷、敌敌畏、敌百虫的耐受力和利用情况。【结果】阴沟肠杆菌培养3h后进入对数生长期,可以利用分解大部分含碳化合物,在营养贫瘠的条件上生长,生长温度范围为10~43℃,生长pH值范围为6~12,对乐果、甲胺磷、敌敌畏、敌百虫的降解利用必须建立在一定的营养物质的基础上。【结论】该菌的繁殖速度快,在营养条件较贫瘠的条件下就可以生存,是一株生存能力很强的菌株,适合应用于土壤、水体环境中的有机磷农药的降解。     

农户对可再生能源沼气选择的影响因素分析——-以江苏省农村家庭户用沼气为例

本研究通过抽样调查法及访谈法对江苏省农村户用沼气消费情况进行了实地调查。数据显示,使用沼气的家庭人均能源消费量低于未使用沼气的家庭,因为沼气对生物质能源有一定的替代作用,减少了秸秆、薪柴的大量粗放使用;同时也有助于能源结构的调整,提高新能源和商品能在家庭用能中所占的比重。对农户是否选择沼气的影响因素分析显示,政府提供资金和技术支持对促进农户选择使用沼气的推动作用最大,人均生猪饲养量、家庭留守人口中最高受教育年限与农户选择行为正相关,人均纯收入、家庭外出打工人口比例、农户位于粮食主产区、农户使用液化气与农户的选择行为负相关。     

马铃薯细胞融合方法的研究

为了完善细胞融合技术,提高融合细胞的分化再生能力,对原生质体的游离、融合、培养条件进行了系统研究,筛选出了既简单又高效率的细胞融合方法多聚鸟氨酸法（poly –L-ornithine, PLO）以及初始、继代、分化培养基,获得了马铃薯花培后代Удача×Жуковский ранний细胞融合再生植株。     

菜豆几丁质酶基因转化马铃薯及后代表达

通过农杆菌侵染和原生质体直接转化法把菜豆几丁质酶基因导入到马铃薯加工品种大西洋中,获得了13株转化后代,经过PCR分析和Southern blot分析,在5株后代中扩增出900bp目标带,说明菜豆几丁质酶基因已整合到马铃薯四倍体基因组中。RT-PCR分析结果表明,其中的3株叶片中有菜豆几丁质酶基因的表达,而且抗病试验进一步证明了该基因在叶片中有较强的表达强度,转基因植株的抗病性比对照提高了30%以上。     

马铃薯可溶性淀粉合成酶SSⅢ基因克隆及生物信息学分析

用RT-PCR方法从马铃薯中克隆了可溶性淀粉合成酶SSⅢ基因的cDNA片段,序列分析表明该cDNA片段全长为3 967 bp,开放阅读框为3 693 bp,编码1 230个氨基酸.核酸序列同源性分析表明,该cDNA与已发表的马铃薯SSⅢ基因(X94400)具有99%同源性,氨基酸序列同源性达98%.应用生物信息学软件对SSⅢ基因分析表明:其编码的蛋白质具有多个磷酸化位点;蛋白质二级结构预测显示,有369个氨基酸可能形成α-螺旋结构,249个氨基酸可能形成β折叠,102个氨基酸可能形成β转角,510个氨基酸可能形成无规则卷曲;通过亚细胞定位可知,它是一种含有75个氨基酸的叶绿体转运肽蛋白.     

茄子花芽分化过程的细胞学观察

本试验以4个不同熟性圆茄品系为材料,采用石蜡切片方法对茄子茎尖分生组织发育的跟踪观察,分析其花芽分化的细胞组织起源、形态变化的过程及不同品种间花发育的差别结果表明：茄子花芽是有主茎或侧枝顶芽分化而来,在茎顸芽向花芽转化时,芽的顶端分生组织由较平滑状转变为向外凸起呈圆锥形;茎顶芽继续分化,分生组织顶部变平增宽,纵切面呈倒梯形,开始形成门茄生殖生长锥,顶芽在分化的过程中其结构符合原套一原体学说;在门茄花芽继续分化的同时,在门茄下部形成两个对生的腋芽,发育成对生的侧枝,证明茄子假二叉分枝的基础是茎顶芽完成了花芽分化;不同基因型的品种进入花芽分化发育进度有明显的差异,早熟品种开花节位低花芽分化相对提前     

利用双杂合位点标记资料构建芒果遗传图谱

为了建立芒果 (MangiferaindicaL )的分子标记遗传图 ,用 15对AFLP (AmplifiedFragmentLengthPolymorphism)引物组合扩增了芒果品种间杂交组合 (Keitt×Tommy Atkins)的 6 0个F1单株 ,获得了 191个多态性位点。它们的分离表现为双杂合 (Aa×Aa)和测交 (Aa×aa)分离两种类型 ,但前者占了 5 9 7%。为了充分利用双杂合位点分离所提供的遗传信息 ,我们根据群体中任意两个双杂合位点隐性个体出现的数目 ,利用二项式分布概率理论推断它们是否连锁以及它们彼此间的相引或相斥关系。在该芒果群体呈 3:1分离的 81个多态性标记中 ,39个被分为 14组 ,以此为基础构建了 15个连锁群 ;这些连锁群共覆盖了 35 4 1cM的芒果基因组。其中 ,最小与最大遗传距离分别为 3 7cM和 2 8 9cM。此外 ,对 18个 1∶1分离类型的标记 ,直接利用Mapmaker作图软件构建了两个芒果连锁群。本文对所提出的利用双杂合位点构建果树遗传图谱的策略进行了讨论。     

芒果种质资源保存研究进展

芒果（Mangifera indica Linn.）是一种重要的热带水果。目前,芒果在全球尚无一个具早熟、高产、优质和抗主要病虫害等优良性状的品种。究其原因：一是大量芒果种质资源由于各种自然和人为原因,以及传统种质保存方法的局限性而逐渐流失;二是由于芒果是基因高度杂合的木本植物,用传统的育种方法改良芒果比较困难。本文对芒果种质资源现状,离体保存技术以及超低温保存技术等方面的研究进展进行了综述,并提出利用体胚再生体系对芒果进行品种改良是未来的一个重要研究方向。     

Numerical taxonomic studies of mango (Mangifera indica L.) varieties in Nigeria

Summary The major characters which account for the variation among the mango varieties in Nigeria were studied using PCA and SCLA. Thirty-one varieties were collected in all ecological zones of the country. Altogether 64 characters were observed and coded for analysis. The results from both methods divided the varieties into two or four groups. The PCA showed that the combination of qualitative and quantitative data as well as combination of few vegetative characters with many reproductive characters are important for mango classification. The primary and secondary characters in classification of mango varieties are discussed.     

Present position regarding breeding of mango (Mangifera indica L.) in India

Summary Progress of hybridization work at various centres in India is reviewed. With the improved technique of mango hybridization and the report of self-incompatibility in mango, it will be possible to evolve a larger number of hybrids for screening for desirable characters. Embryological studies have shown that in mango pollen tubes grow down the style and effect fertilization but the development of zygote is blocked leading to a sporophytic type of self-incompatibility. Hybrids recently evolved through the combinations of regular and commercial biennial bearing varieties indicate that largescale hybridization may offer a solution to the biennial bearing problem in mango.Paper read at the International Horticultural Congress, Maryland, Maryland, U.S.A., 1966.     

芒果MDS家族基因的鉴定、进化及表达分析

2-C-甲基-D-赤藓糖醇-2,4-环焦磷酸合成酶(MDS)是MEP代谢途径中的关键酶.为了鉴定芒果中的MDS家族基因,探索MDS家族基因的进化、表达模式.本研究采用同源搜索的方法,从13个植物基因组中下载了16个MDS家族基因,对其进行了蛋白结构域、系统发育及在芒果中的表达分析.蛋白结构域分析结果显示,芒果MDS家族...     

Effect of minimal processing on bioactive compounds and antioxidant activity of fresh-cut ‘Kent’ mango (Mangifera indica L.)

The influence of dipping in ascorbic acid, citric acid and calcium chloride (AA + CA + CaCl₂) solution and storage time on color, bioactive compounds content and antioxidant activity of fresh-cut mango ‘Kent’ stored at 5 °C was evaluated. The treated mangoes showed better color retention during storage than control mangoes. The dipping treatments with AA + CA + CaCl₂ significantly increased the vitamin C values compared with untreated mango cubes. β-Carotene was not affected by dipping treatments and vitamin E showed a significant decline over storage time for both treated and untreated mango cubes. However, higher vitamin E values were found in treated mangoes. Dipped cubes had higher antioxidant activity measured as TEAC and %RSA than controls. In general, addition of ascorbic acid as an anti-browning agent not only retarded quality loss of fresh-cut mango cubes but also promoted significant increases in antioxidant activity in comparison with control samples.     

红麻GMS与CMS小孢子败育过程的细胞学及组织化学比较

以红麻细胞质雄性不育系(GMS)L23A、保持系L23B及从该保持系中发现的细胞核雄性不育系(CMS)L23BS为材料,采用石蜡显微制片技术,在光学显微镜下比较观察了三者花药发育过程中小孢子的发育过程及组织化学变化。结果表明,L23A的花药发育过程中,小孢子发育的不同阶段均出现败育现象,最早的败育表现为花粉母细胞在减数分裂之前的退化解体,最终形成空的花粉囊; 有的因花粉母细胞减数分裂过程异常而导致不能形成小孢子四分体;有的因小孢子在四分体中不能正常释放而败育,同时绒毡层细胞过度液泡化,并提早解体死亡; 花药发育早期含有少量蛋白质和淀粉,随着花药的发育逐渐变少。而L23BS小孢子败育的时期集中于四分体至单核花粉期间,表现为小孢子发育异常,有些小孢子不能从四分体里释放出来而影响其正常发育。在四分体以前与可育系类似,花药含丰富的蛋白质和淀粉。在败育的过程中,花药中的蛋白质和淀粉含量渐渐减少,但药隔组织中的在颗粒淀粉含量几乎不变。     

杧果种质部分生物学性状指标评价分析

摘要 对杧果树10个主要生物学性状指标主枝与主干夹角、叶片长度、叶片宽度、花序长、花序宽、两性花直径、雄花直径、果实长度、果实宽度、单果重进行了分析,发现其具有丰富的多样性,对包括叶形指数、果形指数在内的12个性状指标进行了分级或分类,提出了现阶段适用性更强的生物学评价参考标准,列出了具体的数据指标,提出了参照品种,为芒果种质资源的系统评价打下了一定基础。     

Evaluation of genetic diversity among commercial cultivars, hybrids and local mango (Mangifera indica L.) genotypes of India using cumulative RAPD and ISSR markers

An assessment of genetic diversity studies was undertaken to understand the level and pattern of diversity in 65 mango (Mangifera indica L.) genotypes of India including 20 commercial cultivars, 18 hybrids, 25 local genotypes and two exotic cultivars based on qualitative and quantitative fruit characters as well as RAPD and ISSR profiles. A considerable variation was observed in respect of three important qualitative characters namely table quality, fruit attractiveness and storage life of ripe fruits and potentially superior genotypes for the above traits were identified. A wide variation was noticeable regarding metabolite composition of pulp of ripe mango fruit with respect to total soluble solids, total sugar, reducing sugar, acidity, sugar:acid ratio, ascorbic acid and phenolic content. Fifteen RAPD primers yielded 27 monomorphic and 129 polymorphic bands with percent polymorphism averaging 82.7%. Of a total 70 ISSR bands generated from eight ISSR primers, 60 bands (85.71%) were found to be polymorphic. Cumulative band data from these two methods precisely arranged accessions into eight clusters which correspond well with their pedigree relationship. UPGMA dendrograms drawn using RAPD, ISSR and cumulative data showed highly similar grouping of genotypes on the basis of their parental origin. No clear-cut geographical separation was revealed among East, West, North and South Indian mango cultivars by neither of these molecular markers nor their combinations. This supports the common gene pool origin of mango as well as operation of similar selection pressure as the cultivar preferences in these areas are largely similar.     

Development of CAPS markers and their application in breeding for mango, Mangifera indica L.

To develop cleaved amplified polymorphic sequence (CAPS) markers for the identification of true hybrids in F1 progeny, we cloned sucrose synthase (SS) and sucrose phosphate synthase (SPS) genes from 19 mango cultivars. Through comparison of amino acid sequences in a BLASTX search, cloned SS and SPS genes were found to be homologs of Citrus unshiu CitSUS1 (AB022092) and CitSPS1 (O22060), respectively. Therefore, we designated the SS and SPS genes as MiSUS1 and MiSPS1, respectively. There were 11 genotypes in MiSUS1 and 10 in MiSPS1 sequences. In the MiSUS1 nucleotide sequences, 46 SNPs and 5 in/dels were detected. Twenty-six out of the 46 SNPs mapped on the restriction enzyme recognition sites were successfully converted to 25 CAPS markers. In the MiSPS1 nucleotide sequences, 99 SNPs and 6 in/dels were detected. Thirty out of 99 SNPs and 2 out of 6 in/dels mapped on the recognition sites were also converted to 32 CAPS markers. By using two CAPS markers, SS-2798 and SPS-5745, we identified 37 true hybrids that had different genotypes to ‘Irwin’ from 82 F1 progeny obtained through a cross between ‘Irwin’, the maternal parent, and paternal parents by using flies as pollinators. The method developed here to identify true hybrids is a simple, rapid and highly reliable tool for efficient mango breeding.