以cosmid质粒为载体的稻瘟病菌转化体系的建立及病菌基因文库的构建

 用潮霉素B（ hygromycin B）抗性基因和λ噬菌体的cos位点组建了一种cosmid质粒pSVhygB，以此质粒建立了稻瘟病菌的转化体系，并且构建了病菌的基因组文库。结果表明，pSVhygB质粒可用于稳定地转化稻瘟病菌，转化频率约为15个转化子／μg DNA，电击法转化并不能有效地提高稻瘟病菌的转化频率。构建的稻瘟病基因文库中包含将近10000个重组子克隆，随机挑取12个重组子的鉴定表明，都包含有外源插入片段，说明所建文库已达要求。     

抗真菌蛋白Rs—AFPs基因克隆与序列分析

从开花后５０－９０ｄ的萝卜种子中提取总ＲＮＡ,逆转录合成ｃＤＮＡ第一链,通过ＰＣＲ反应,得到Ｒｓ－ＡＦＰ１和Ｒｓ－ＡＦＰ２基因扩增产物。采用Ｔ－载体克隆技术,将二分别插入ｐＢｌｕｅｓｃｒｉｐｔ／ＥｃｏＲＶ－Ｔ克隆载体,得到重组质粒ｐＧＲ－１和ｐＧＲ－２用其转化宿主菌Ｅ．ｃｏｌｉ ＤＨ５α,从阳性菌落制备测序用质粒ＤＮＡ样品,在ＡＢ１３７０Ａ ＤＮＡ测序仪上测序。     

花生条纹病毒外壳蛋白基因在大肠杆菌中的表达

用内切酶Ｂａｍ ＨＩ和ＥｃｏＲＩ将ｐＧＥＭ－ ＳｔＶ 含有的约１１Ｋｂ 大小的ＰＳｔＶ－ ｃｐ 基因切下定向插入到表达质粒ｐＢＶ２２０ 的启动子ＰＲ、ＰＬ的下游,构建了该基因的原核表达载体ｐＢＶ－ ＳｔＶ。ＳＤＳ－ ＰＡＧＥ凝胶电泳结果表明：ＰＳｔＶ－ ｃｐ 基因在大肠杆菌ＤＨ５α中经温度（４０℃）诱导后,可特异地表达３３ｋＤ蛋白。     

人α—干扰素2b基因转化番茄,番木瓜的研究（I）：人α—干扰素2b基因？…

以ＰＢＶ８８９（α－ＩＦＮ－２ｂ）为基因供体,利用ＥｃｏＥ Ｉ／Ｐｓｔ Ｉ双酶切分离出基因α－ＩＦＮ－２ｂ,再利用现有的植物表达质粒ＲＯＫ Ⅱ（带ＰＲＶ－ＣＰ基因,３５Ｓｐｒｏｍｏｔｅｒ／ＮＰＴ Ⅱ）,用ＸｂａＩ／ＳａｃＩ双酶切分离出大片段（Ｅ６）作为基因受体,采用定向连接法将α－ＩＦＮ－２ｂ拼接在Ｅ６上,完成植物表达载体构建。采用ＤＡＮ ｄｏｔ ｂｌｏｔ、ＲＮＡ ｄｏｔ ｂｌｏｔ对重组子进行筛选     

抗菌肽B基因的点突变及在昆虫细胞中的表达

抗菌肽Ｂ基因经ＰＣＲ定点突变技术改造后,克隆到杆状病毒转移载体ｐＡｃＧＰ６７Ｂ中的信号肽位点上,随后与野生ＡｃＮＰＶ－ＤＮＡ共转染Ｓｆ９细胞,经空斑筛选、ＰＣＲ检测证实获得含该基因的重组病毒。重组病毒在Ｓｆ９细胞中扩增并表达抗菌肽Ｂ基因。取含表达产物的细胞培养液检测其生物活性表明：改造过的抗菌肽Ｂ基因在Ｓｆ９细胞中获得初步表达,且表达的分泌型产物对ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＫ１２Ｄ３１有抑菌活性。     

提高稻瘟病菌转化频率的研究

 为提高pAN7-1质粒导入稻瘟病菌建立的遗传转化体系的转化率，进行了对转化效率影响因子的研究。稻瘟病菌转化溶液中使用50 mmol/L CaCl₂浓度，PEG4000 40%或PEG80000 40%多聚物，添加30 μg鲱鱼精DNA，供体质粒DNA呈直线状都有利转化频率的提高。其中鲱鱼精DNA作用尤为显著，由于添加30 μg鲱鱼精DNA，转化频率高达71.6转化子/μg DNA。42℃热冲击对稻瘟病菌转化并非必需，转化溶液中添加受体菌2539w DNA后，反而引起转化频率的明显下降。     

玉灵菌茶研究初报

玉灵菌，即茯苓菌，属于担子菌纲（Ｂａｓｉｄｉｏｍｙｃｅｔｅｓ）多孔菌目（Ｐｏｌｙｐｏｒａｌｅｓ）多孔菌科（Ｐｏｌｙｏｉｒａｃｅａｅ）卧孔属（Ｐｏｒｉａ）学名为Ｐｏｒｉａ ｃｏｃｏｓ（Ｓｃｈｗ）Ｗｏｌｆ．玉灵菌茶，即以茶叶为主要基质，配其他食药兼融的材料，组合成培养基，接入功能性茯苓真菌，经过人工调控培养，加工培养基而形成的生物菌体茶。现根据作者多年研究该茵在茶基质上的生长规律及相关加工工艺等方面结果，阐述如下： １培养基配制 从茶叶的物质组成成份来看，茶叶富含醇类、醛类、多酚类以及无机物Ｉ．Ｓ等抑…     

香蕉ACC合成酶含3''''末端的cDNA克隆

采用 ３＇ＲＡＣＥ方法对香蕉（Ｍｕｓａ ＡＡＡ Ｃａｖｅｎｄｉｓｈ Ｓｕｂｇｒｏｕｐ）ＡＣＣ合成酸含 ３＇末端的 ｃＤＮＡ进行扩增，扩增产物被克隆到ｐＣＲ■２．１载体上，转化大肠杆菌ＤＨ５ α，筛选到重组质粒（ｐＡＣＳ２），并对插入片段进行序列测定。结果表明，３＇ＲＡＣＥ产物长 １６８０ ｂｐ，其中一个 开放读框内的核苷酸序列编码４４４个氨基酸，氨基酸序列包括了ＡＣＣ合成酶中所应具有的７个高度保守区。同时该产物还有长２６９ ｂｐ的３＇末端，并具有完整的 Ｐｏｌｙ（Ａ）尾（２４ｍｅｒ）。     

利用显性选择标记基因转化稻瘟病菌（英文）

 利用抗药性标记基因(Hygromycin B, hygB)建立稻瘟病菌的转化系统。结果表明， Aspergillus的TrpC转录信号与细菌的hygB抗性结构基因重组的质粒pCSN43可取在稻瘟病菌中表达，该质粒与受体菌无同源顺序，因而有利于对转化菌株进行筛选和分析。 电激法和PEG/Ca^2＋ 法均适用于转化稻瘟病菌，但后者的转化效率比前者高。     

双价抗菌肽基因转化辣椒

在建立高效快速辣椒（Ｃａｐｓｉｃｕｍ ａｎｎｕｕｍ Ｌ．）子叶离体培养和植株再生体系的基础上,将昆虫抗菌肽Ｂ、Ｄ基因构建而成的双价质粒ｐＣＤＢ－Ⅱ以逐杆菌为介导转入５个辣椒栽培品种,共获得Ｋａｎ＾Ｒ植株１２００多株,对部分Ｋａｎ＾Ｒ植株进行点杂交、ＰＣＲ、Ｓｏｕｔｈｅｒｎ杂交检测,结果证明了外源基因被成功整合。盆栽接青枯菌试验,结果显示,转基因植株具有较强的抗病力。     

Analysis of nutritional components of eight famine foods of the Republic of Niger

In the western Sahel, indigenous plants become important staples when cereal harvests are inadequate to support populations inhabiting that region of Africa. The purpose of this study was to assess the nutrient content of several of these edible wild plants. The leaves of the following seven plant foods were analyzed: Ziziphus mauritiana, Cerathotheca sesamoides, Moringa oleifera, Leptadenia hastata, Hibiscus sabdarifa, Amaranthus viridi, and Adansonia digitata. The fatty acid, vitamin E, carotenoid, selected mineral and amino acid contents of these plant foods were determined. These same analyses were performed on the fruit of the Adansonia digitata. In quantitative and qualitative terms, Amaranthus viridis was found to be an excellent source of protein. Its amino acid composition compared favorably to that of a World Health Organization (WHO) protein standard. It also contained considerable amounts of the two fatty acids that are essential in humans (linoleic and -linolenic) and a number of minerals including iron, magnesium, calcium and zinc. The leaves of Hibiscus sabdarifa contained an appreciable quantity of protein the composition of which was comparable to the WHO standard. The mineral content of the leaves of this plant was also exceptionally high; noteworthy was its high zinc content. H. sabdarifa also contained significant quantities of the two essential fatty acids. Ziziphus mauritiana was an excellent source of the essential fatty acid linoleic acid and several of the metals including iron, calcium, magnesium and zinc. Its content of other essential nutrients, however, was rather low. In general, Adansonia digitata leaves were nutritionally superior to the fruit of the tree; however, the fruit did contain useful quantities of potassium, phosphorus, zinc and -linolenic acid. The Leptadenia hastata leaves were an especially good source of lutein and -carotene. These data should be useful to the people who inhabit the western Sahel in helping them devise healthy diets during times when cereal staples are in short supply.     

狗牙根诱变后代抗寒性评价

通过电解质外渗法和匍匐茎恢复试验对‘阳江’狗牙根及其12个通过形态鉴定选出的坪用价值高且花序密度低的诱变后代进行抗寒性鉴定。电解质外渗法结果表明:诱变后代间的抗寒性具有较大差异,其叶片半致死温度(LT₅₀)的变异范围为-7.6～-0.2℃(最低值与最大值相差7.4℃);参试材料抗寒性由强到弱依次为M18>M4>M26>M28> M22>阳江>M29>M31>M10>M37>M16>M1>M25,其中,有5个诱变后代抗寒性优于亲本,分别是M18、M4、M26、M28、M22。匍匐茎恢复实验结果表明:诱变后代M1、M22、M26、M31、M25在0℃和-5℃低温胁迫后的恢复生长率都高于亲本,恢复能力均优于亲本;M10、M37、M28在-5℃低温胁迫下,恢复生长能力低于亲本,抗寒性相对较弱,M16和M4在0℃和-5℃低温胁迫下,恢复生长率都低于亲本,抗寒性明显弱于亲本。综合2种方法鉴定结果显示:诱变后代M1、M25的恢复能力较强;M4、M28的叶片抗寒性较好,青绿期较长;M22、M18、M26的叶片抗寒性和匍匐茎恢复能力均较强;M29,M31的叶片抗寒性和匍匐茎恢复能力与亲本相似;M10、M16、M37的叶片抗寒性和匍匐茎恢复能力均较弱,整体抗寒性较弱。     

芦笋枯萎病病原鉴定

对造成福建省漳州市东山县的芦笋枯萎病病原菌进行分子鉴定,以期确定病原菌的属、种名,为防治该病害寻求理论依据。试验对分离纯化的病原菌核糖体DNA的ITS区进行测序,在Genbank中搜索其同源性并构建它们的系统发育树,结果表明：引起芦笋枯萎病的病原菌是镰刀属中的尖孢镰刀菌(Fusarium oxysporum),且病原菌之间的同源性高达89%,遗传差异不显著。     

橡胶树丛枝病病原的抗血清制备与应用

以感染橡胶树丛枝病病原的长春花为材料,制备得到橡胶丛枝病病原菌抽提液,以抽提液为抗原免疫家兔,制备抗血清,经微量沉淀测定,抗血清效价为１：２０４８。应用橡胶丛枝病抗血清检测橡胶褐皮病,无症苗木检出率达３０％－３７％,可疑的褐皮病树检出率达８５．７％。     

Size and frequency of occurrence of pre-roost gatherings of starlings

One hundred and fifty-six pre-roost gatherings of starlings were observed at 28 sites around a roost in west Norfolk during winter. Pre-roost gatherings occurred more frequently at individual farmyards compared with individual fields, but 82% of the sites where gatherings occurred were on fields. The size of gatherings was greater, the closer to the roost. Birds in pre-roost gatherings on fields of autumn-sown winter wheat spent most of the time feeding and had a diet composed almost exclusively of wheat seeds. Birds in gatherings on grass fields also fed intensively whereas only 31% of birds at farmyards were feeding. The implications that these results have with regard to potential damage to fields of winter wheat and around farmyards are discussed.     

农杆菌注射法瞬时转化荔枝果实组织的研究

为了寻找一种高效的荔枝果实瞬时基因表达方法,本研究以荔枝品种‘新球蜜荔’（Litchi chinensis Sonn. var. ‘Xinqiumili’）为试材,利用农杆菌注射法对荔枝果实组织进行转化,研究了果实发育时期、菌株种类、注射部位、取样时间、菌液浓度等对转化效率的影响。结果表明：选择果肉已完全包裹种子的Ⅱb期果实进行连体注射,在果柄、果皮、种子、果肉分别注入OD₆₀₀值为2.4的农杆菌菌株GV3101,4 d后取样进行检测,4个组织的GUS染色率较高。本研究成功建立了适用于荔枝果实的基因瞬时表达系统,为今后快速鉴定荔枝果实相关基因功能提供了基础。     

《生物安全法》实施背景下基因编辑技术的安全评价与监管

2020年10月17日,《中华人民共和国生物安全法》(以下简称《生物安全法》)通过全国人大常委会的审议,自2021年4月15日起施行.生物技术的研究与应用安全是《生物安全法》涉及的主要内容之一,基因编辑作为近几年生物技术领域的研究热点,其安全性评价和监管备受关注.本文概述了基因编辑技术的应用现状,比较了不同国家对基因编...     

蝉拟青霉疏水蛋白PChyd基因表达模式分析及敲除载体构建

为深入研究昆虫病原真菌蝉拟青霉疏水蛋白PChyd基因的功能,根据蝉拟青霉基因组信息克隆疏水蛋白PChyd基因,对该基因序列进行生物信息学分析,使用qRT-PCR技术对其在不同培养条件或阶段下的表达模式进行分析,并通过酶切酶连的方法构建了该基因的敲除载体。结果表明：PChyd基因的开放阅读框序列全长303 bp,编码100 aa,包含22 aa的信号肽序列和70 aa疏水蛋白功能区域。系统发育分析显示该基因与粗糙虫草菌亲缘关系最近。qRT-PCR结果显示PChyd基因在PDA培养的菌丝体、诱导的附着胞、诱导的芽生孢子中表达量显著高于另外2个样品,其中芽生孢子表达量最高,暗示该基因在蝉拟青霉侵染初期和在昆虫血腔中定殖阶段可能具有重要作用。凝胶电泳结果表明,成功构建了该基因的敲除载体,扩增出含有上臂、HPH、下臂的3356 bp左右的片段。本研究为进一步探究蝉拟青霉疏水蛋白PChyd基因的致病机理、生防工程菌的改造奠定了基础。     

波罗蜜叶片突变体叶绿素含量测定和超微结构观察

波罗蜜（Artocarpus heterophyllus）是一种热带果树,至今对其突变体的研究少有报道。本研究以波罗蜜叶片为试验材料,探究其叶片出现白化和返绿现象的可能原因。（1）用分光光度计法、比色法测定波罗蜜叶片突变体和正常绿叶的叶绿素及叶绿素前体物质含量;（2）用叶绿素酶Elisa试剂盒测定叶绿素酶活性;（3）用透射电子显微镜对叶绿体的超微形态结构进行观察。研究结果表明：（1）不同叶色所产生的叶绿素a含量、叶绿素b含量、叶绿素总含量、类胡萝卜素含量均存在显著差异;（2）波罗蜜叶片突变体和正常绿叶的叶绿素合成前体物质含量之间并没有出现显著性差异;（3）对波罗蜜叶片的叶绿素酶活性进行测定时,发现其活性出现显著差异,但对其叶绿素含量的差异性并没有产生较大影响;（4）观察波罗蜜叶片内叶绿体超微形态结构时,发现正常绿叶的叶绿体形态完好且数量较多,白化叶和返绿叶的叶绿体内部结构存在缺陷,其原因是叶绿体基粒构建阶段受阻;基于测定波罗蜜叶片中的叶绿素、叶绿素前体物质含量和叶绿素酶活性,并观察波罗蜜叶片内叶绿体的超微形态结构,对得到的数据结果进行比较分析。本研究推测是在叶绿素合成阶段,原脱植基叶绿素合成叶绿素时受阻及叶绿体发不良导致波罗蜜出现白化和返绿现象,为今后进一步综合研究波罗蜜突变体提供理论依据。     

Mechanism of action of insecticidal secondary metabolites of plant origin

Insect pest management is facing the economic and ecological challenge worldwide due to the human and environmental hazards caused by majority of the synthetic pesticide chemicals. Identification of novel effective insecticidal compounds is essential to combat increasing resistance rates. Botanicals containing active insecticidal phytochemicals appear to be promising to address some of these problems. Therefore, there is a continuous need to explore new active molecules with different mechanisms of action. Secondary metabolites present in plants apparently function as defense (toxic), which inhibits reproduction and other processes. The phytochemical biomolecules could be used for maximizing the effectiveness and specificity in future insecticide design with specific or multiple target sites, while ensuring the economic and ecological sustainability. In this article, the current state of knowledge on phytochemical sources and insecticidal activity, their mechanism of action in insects, resistance, and promising advances made in phytochemical research are reviewed.