水稻白叶枯病菌GX1329基因组文库的构建及含编码TAL效应物基因的克隆的分离

由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50％以上。研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物。为了解水稻白叶枯病菌广西菌株GX1329中含有avrBs3／pthA家族基因的情况,本研究应用Alu I部分酶切其基因组DNA,构建了含有736个克隆的菌株GX1329的基因组文库。BamHI酶切分析随机挑取的15个文库克隆表明,克隆的外源DNA随机性良好,克隆的最小片段为27．7kb,最大为58．5kb,平均大小为39．9kb,文库克隆容量约为2．8×10^3Mb,该文库中包含基因组中任一个基因的概率为99．4％。利用来自水稻白叶枯病菌菲律宾菌株PX086的无毒基因avrXa10的第252位～第486位核苷酸序列作为探针,通过菌落原位杂交从GX1329基因组文库中筛选到37个含avrBs3／pthA家族基因的克隆。再通过Southern杂交分析,得到了17个独立克隆。这17个克隆中至少含有13个不同的avrBs3／pthA家族基因。这些基因在GX1329基因组中有的单独存在,有的两个或两个以上串联存在。本工作基本上明确了菌株GX1329基因组中avrBs3／pthA家族基因的数量,为进一步研究菌株GX1329中avrBs3／pthA家族基因的功能奠定了基础。     

设施菜地土壤次生盐渍化分类与分级标准

近三十年来,我国设施蔬菜栽培发展迅速,已成为我国农业中最有活力的新兴产业之一。但在长期的设施蔬菜生产过程中,普遍存在着不合理施肥、养分比例失调、肥效下降和资源浪费严重等问题,致使土壤养分富集,盐分积累现象日趋严重。本文对我国设施菜地土壤次生盐渍化的现状、发生原因及其对作物的危害进行综述和分析,在此基础上制定设施菜地土壤次生盐渍化分类与分级的标准的建议及初步构架,旨在为防治设施菜地土壤次生盐渍化,实现设施菜地土壤的质量调控与可持续利用提供理论指导和科学依据。     

黄瓜果实苦味(Bt)基因的插入缺失(Indel)标记

为了筛选与黄瓜果实苦味(Bt)基因紧密连锁的插入缺失(Indel)标记,本研究以黄瓜(Cucumis sativusL.)果实苦味和果实不苦的高代纯合自交系46GBt和931为亲本构建的F2分离群体为试材,明确了黄瓜果实苦味性状遗传是符合单显性基因控制的质量性状.同时在完成Bt基因初步定位和双亲重测序的基础上,利用生物信息学分析双亲在初步定位区域的序列差异,设计了7对InDel标记.通过对含有184个单株的F2群体分析,获得了与Bt基因连锁距离为0.8 cM的Indel标记Bt-InDel-1.利用不同于本研究且包含58个单株的黄瓜F2群体材料对Bt-InDel-1的验证分析表明,该标记检测的正确率达到94.8％.本研究结果可用于无苦味黄瓜的分子标记辅助选择(MAS)育种及Bt基因精细定位.     

彩色马蹄莲2n花粉诱导及其三倍体植株的获得

2n配子介导的有性多倍化因其具有染色体重组创造新型变异的机会而越来越受到育种家的重视.本研究用化学试剂秋水仙素处理彩色马蹄莲(coloured Zantedeschia hybrid)二倍体栽培种幼蕾诱导2n配子,并对2n配子形成的细胞学特征进行了显微观察.结果显示,秋水仙素处理可以诱导彩色马蹄莲2n花粉产生,2n花粉的花粉粒明显变大,适宜的秋水仙素处理浓度为0.15％;配子形成过程中出现平行、融合等纺锤体定位异常现象,四分体时期出现二分体、三分体,产生减数分裂异常的多核细胞、小孢子形成过程中核融合等异常现象;诱导的2n花粉与正常1n雌配子杂交,杂交后代经染色体鉴定筛选获得三倍体后代(2n=3x=48).本研究结果表明,人工诱导2n花粉是培育彩色马蹄莲有性多倍体的一种有效可行的方法.     

通过转基因手段改善作物产量性状

在世界范围内,目前的作物转基因研究主要针对于提高作物自身的抗逆性开展,而产量是一个较为复杂的农艺性状,受多种基因与环境的相互作用所控制.其中,光合作用、氮素同化、碳源分配、植株株型等生理过程是产量形成的基础.随着植物基因组测序工程的逐渐完善,控制上述生理过程的许多基因已被成功分离,并通过基因工程手段对其进行遗传修饰,最终获得了产量性状得以明显改善的转基因植物.虽然目前对于基因如何调控每个性状的机制尚未得到充分阐明,但是这些基因的获得及其增产功能的验证为作物的高产育种提供了一条全新的思路和一系列新方法.随着国家转基因生物新品种培育科技重大专项的实施,对功能基因的认识显得尤为重要,本文主要从影响作物产量形成的源、流、库、株型、氮素及水分利用率等几方面入手,对近10年来控制植物产量性状基因的相关研究进行了综述,期望为高产转基因作物新品种的培育提供一定的理论依据.     

Novel royal jelly proteins identified by gel-based and gel-free proteomics

Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.     

Comparison of the chemical constituents of aged pu-erh tea, ripened pu-erh tea, and other teas using HPLC-DAD-ESI-MSn

Pu-erh tea is a popular beverage in southwestern China and South Asian countries. To explain the differences of aged pu-erh tea and ripened pu-erh tea, the chemical constituents of these teas were identified by HPLC-DAD-ESI-MS(n). In addition, HPLC was used to determine the contents of the major polyphenols, gallic acid, caffeine, and theobromine, in various types of teas. These results showed that the majority of chemical constituents in ripened pu-erh tea and aged pu-erh tea were similar, but the contents of catechins and gallic acid presented significant differences between these two teas. After fermentation by microorganism, the levels of catechins in ripened pu-erh tea were decreased, but the contents of gallic acid and caffeine were conversely elevated compared with aged pu-erh tea.     

Detachment of road surface soil by flowing water

An agricultural watershed generally consists of two land use categories, farmland and the unpaved road or path networks with different traffic frequency and size. Road surfaces are quite different from farmland soil in physical properties, resulting in it's distinguish production transportation process. Hydraulic flume experiments were conducted with the flow discharges ranging from 1 to 5 L s^− 1 and the slope gradients ranging from 8.8% to 46.6% to simulate the soil detachment process on a road surface and to develop tools in order to calculate detachment rates occurring on that road surfaces. The results illustrate that road surfaces behave characteristically in the runoff detachment and sediment delivery process due to the difference in the bulk density and functions of agricultural soils. The soil detachment rate is closely related to flow depth, slope gradient and other hydraulic parameters such as shear stress, stream power and unit stream power. Multiple non-linear regression analyses indicate that detachment rates for all roads can be accurately predicted by power functions of flow depth and slope gradient. According to the experimental results, stream power was suggested as an indicator to estimate soil detachment rate instead of shear stress in soil erosion models. However, considering the simplicity and availability, power function of flow depth and slope gradient is also recommended to predict detachment rate on the road surfaces.     

人工合成抗菌肽D2A21基因的克隆和表达

为了获得高效表达的抗菌肽D2A21,本研究根据抗菌肽D2A21的氨基酸序列及大肠杆菌偏爱的密码子,设计并人工合成D2A21基因,并将该合成基因克隆至pProEXHTb载体中,构建成含有含6个组氨酸标签的重组质粒。重组质粒转化至大肠杆菌中,经摇瓶发酵,IPTG诱导后,发酵液用聚丙烯酰胺凝胶电泳分析。结果表明,成功合成D2A21基因,并构建了抗菌肽D2A21表达载体。通过镍柱亲和层析纯化,聚丙烯酰胺凝胶电泳分析,抗菌肽D2A21能够在大肠杆菌中稳定表达。本研究将为大规模表达纯化D2A21及其进一步的研究和利用提供一定基础。     

植物EST-SSR标记开发及其应用

随着生物科技的进步,大量的植物表达序列标签（expressed sequence tags,ESTs）已经成为开发SSR标记的重要资源。EST-SSR作为一种新型分子标记,其多态性可能与基因功能直接相关,而且在相近植物间具有良好通用性,使得EST-SSR标记在实际应用中更具价值。采用计算机方法大规模发掘EST-SSR多态性位点极大地提高了SSR标记开发效率。尤其随着新一代测序技术的成熟以及测序成本的急剧下降,利用新一代测序技术产生大量的转录组数据进行EST-SSR多态性标记的计算机大规模发掘将对SSR标记的开发带来深远影响。本文简要介绍了植物EST-SSR分布特点,并对EST-SSR标记开发现状以及相关应用作了综合评述,此外,还对EST-SSR标记的计算机开发新策略进行了展望。