家蚕IAP互作蛋白的筛选、鉴定及其对BmNPV增殖的影响

目的 细胞凋亡作为昆虫免疫应答的重要组成部分,在病毒与宿主相互作用的过程中扮演着重要角色,以细胞凋亡相关基因为研究对象对阐明其在病毒感染过程中的作用具有重要的参考价值。本研究旨在筛选家蚕凋亡抑制蛋白（Bombyx mori inhibitor of apoptosis protein,BmIAP）的相互作用蛋白,并验证其在家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus, BmNPV）侵染过程中与Bmiap的相互调控关系及在BmNPV增殖过程中的作用,为探明宿主与BmNPV相互作用的分子机制提供理论依据。方法 利用免疫共沉淀技术筛选在BmNPV侵染家蚕细胞过程中与BmIAP相互作用的蛋白,对获得的特异性差异条带进行LC-MS/MS蛋白质谱分析,根据蛋白分子量大小并利用生物信息学方法对获得的蛋白进行鉴定,确定候选基因;通过分子克隆技术对候选基因进行克隆,利用SMART在线工具及BioEdit分别对候选基因的结构域进行预测及多序列比对分析;通过免疫荧光试验验证BmIAP和候选蛋白的细胞共定位情况,并进一步利用免疫共沉淀验证它们的相互作用;分别通过真核表达和基于CRISPR/Cas9的基因编辑系统对候选基因进行过表达和敲除,利用qRT-PCR技术检测相应基因的表达情况,以确定Bmiap和Bmpp5在BmNPV侵染过程中的调控关系;同样,在过表达和敲除Bmpp5后检测杆状病毒Vp39的表达量,以确定Bmpp5对BmNPV增殖的影响。结果 通过免疫共沉淀获得7个可能与BmIAP相互作用的宿主蛋白和1个BmNPV蛋白,进一步分析确定1个与凋亡相关的候选基因Bmpp5;Bmpp5的开放阅读框为1 473 bp,编码490个氨基酸,预测的蛋白分子量约为56 kD,含3个TRP结构域和1个PP2Ac结构域,在昆虫间具有较高的保守性;免疫荧光检测证明BmIAP和BmPP5共定位于细胞质中,免疫共沉淀结果表明两者可以相互作用;在BmNPV侵染过程中,过表达Bmiap后,Bmpp5的表达量显著上调,而敲除Bmiap后,Bmpp5的表达量显著下调,表明Bmiap能够促进Bmpp5的表达;同时,过表达Bmpp5后,Bmiap显著上调表达,而敲除Bmpp5后,Bmiap显著下调表达,表明Bmpp5同样能够促进Bmiap的表达;过表达Bmpp5能够促进BmNPV的增殖,敲除Bmpp5能够抑制BmNPV的增殖,表明Bmpp5的表达利于BmNPV的增殖。结论 鉴定了1个能够与BmIAP相互作用的蛋白BmPP5,且该蛋白在昆虫中高度保守;在BmNPV侵染过程中,Bmiap和Bmpp5能够相互促进,且Bmpp5能够促进BmNPV的复制增殖。     

马铃薯低温响应的ScmiR390-5p及其靶基因表达与结构分析

【目的】研究马铃薯富含亮氨酸重复序列(leucine-rich repeat receptor-like protein kinase LRR-RLK)类受体蛋白激酶基因SCLRRK1受miR390（ScmiR390-5p）调控响应非生物胁迫的机理。【方法】通过低温响应马铃薯miRNA测序分析与靶基因预测,发现ScmiR390-5p通过调控1个可能的LRR类受体蛋白激酶基因对低温做出响应;利用实时荧光定量PCR技术（Quantitative Real-time PCR,RT-qPCR）验证ScmiR390-5p和SCLRRK1响应低温胁迫的表达情况;利用RLM-5′RACE确定ScmiR390-5p作用于SCLRRK1的切割位点;利用PCR技术克隆得到SCLRRK1的DNA序列和CDS序列,生物信息学分析SCLRRK1的结构和功能;利用RT-qPCR分析ScmiR390-5p/SCLRRK1在马铃薯各组织中的表达情况及其响应各种非生物胁迫的表达情况。【结果】RT-qPCR结果表明,低温诱导ScmiR390-5p表达,SCLRRK1的表达则受到低温的抑制,ScmiR390-5p和SCLRRK1的表达在低温胁迫下呈负相关性;RLM-5′RACE分析表明,ScmiR390-5p作用于SCLRRK1的切割位点是ATTCCT//CCTGAGTT,马铃薯ScmiR390-5p/SCLRRK1调控模块在低温响应中起作用。克隆结果表明SCLRRK1的CDS序列长度为2 685 bp,编码894个氨基酸,DNA序列长度为3 549 bp,含有1个内含子、2个外显子、3′非编码区和5′非编码区,ScmiR390-5p的靶位点位于SCLRRK1 CDS序列内部（960—981 bp,GGAACTATTCCTCCTGAGTTT）。生物信息学显示SCLRRK1是1个富含亮氨酸重复序列(leucine-richrepeat,LRR)的类受体蛋白激酶基因,编码的蛋白属于跨膜分泌蛋白。马铃薯组织表达RT-qPCR分析显示,ScmiR390-5p在叶中的表达量最高,根次之,茎中（地上茎、块茎和匍匐茎）表达量较低;而SCLRRK1的表达情况与ScmiR390-5p相反,其在叶中的表达最低,在茎中表达最高（匍匐茎>块茎>地上茎）。多种非生物胁迫结果显示,ScmiR390-5p和SCLRRK1表达在NaCl胁迫下呈负相关,ScmiR390-5p受到NaCl胁迫诱导表达。与对照相比,ABA和6-BA处理下ScmiR390-5p表达先下降之后呈波浪小幅回调;6-BA处理下SCLRRK1表达持续升高,ABA处理下SCLRRK1表达先上升后下降。GA₃、PEG和IAA处理8 h时,ScmiR390-5p的表达达到峰值,GA₃、PEG和IAA处理都能诱导SCLRRK1表达,但其变化趋势与ScmiR390-5p相关性不强。【结论】SCLRRK1具备编码LRR类受体蛋白激酶的核酸、氨基酸序列和结构基础,是ScmiR390-5p的靶基因;ScmiR390-5p/SCLRRK1调控模块在马铃薯组织器官中具备明显作用;ScmiR390-5p在转录后水平通过抑制马铃薯SCLRRK1的表达对低温胁迫做出响应,同时,马铃薯ScmiR390-5p/SCLRRK1调控模块在NaCl和6-BA胁迫响应中起作用,但不对ABA、GA₃、IAA和PEG胁迫做出响应,ScmiR390-5p和SCLRRK1分别在转录水平受到ABA、GA₃、IAA和PEG胁迫信号调控。     

Stem elongation in Pennisetum purpureum results from a fixed pattern of vegetative development potentially enhanced by the initiation of flowering

The characterization of stem elongation is of fundamental importance in C4 tropical grasses as it affects forage quality and determines optimal management practices. The objectives of this study were to analyse the determinants of stem elongation and leaf area production in shoots of Pennisetum purpureum cv. Napier (elephant grass) using unstressed isolated plants. Three experiments were conducted in Brazil during the spring, summer and autumn seasons. Regular measurements of leaf and pseudostem length were performed on the main and primary axes. Ten destructive measurements were also performed during each experiment to monitor apical meristem height, internode length and the number of initiated leaves. The onset of stem elongation occurred at the same vegetative stage (i.e., appearance of leaf 13) irrespective of the seasons and experiments. The first internode to elongate belonged to phytomer 8, and a constant lag of five phyllochrons was systematically observed between internode production and its rapid elongation period. Higher stem and internode elongation rates were observed during the reproductive phase (autumn) versus the vegetative phase (summer and spring group). Maximal internode length reached 8–10 cm in summer and spring and 20 cm in autumn, at approximately phytomers 12–13. A similar pattern was reported for all primary axes irrespective of the experiments, the position of the first internode to elongate descending regularly down the main axis. These results provide key elements to predict the onset of stem elongation in the field from simple measurements. They could contribute to improving crop models for perennial tropical C4 grasses.     

Phenotypic Analysis and Gene Cloning of Rice Floury Endosperm Mutant fse4

【Objective】Starch is the main energy reserve of rice endosperm. The biosynthesis of starch is complex, requiring a large number of synthetic enzymes and regulators. Screening rice endosperm defective mutants and cloning the underlying genes will lay theoretical basis for starch biosynthesis and its regulation. 【Method】 A stable genetic floury and shrunken endosperm mutant termed as fse4 (floury and shrunken4) were obtained from the mutant library of Ningjing 3 (WT), which was induced by N-methyl-N-nitrosourea (MNU). An F2 mapping population was generated by crossing the fse4 mutant with Dular (an indica rice variety) and the gene was finally isolated. The floury seeds segregated from the fse4 heterozygous plants were used to observe the morphological features, and the physicochemical properties of the brown rice flour were analyzed. The endosperm structure was observed with a scanning electron microscopy by the semi-thin section technology. The expression of starch synthesis related genes during grain filling was determined by qRT-PCR; Immunoblotting was used to detect the accumulation of proteins related to starch synthesis. The amino acids contents of each mature endosperm were determined with the fully automatic amino acid analyzer.【Result】The 1000-grain weight and grain size were significantly reduced in fse4. Compared with WT, the contents of total starch, amylose and total protein were signi?cantly lower in fse4, while the lipid content was signi?cantly higher. The starch viscosity, breakdown viscosity and setback viscosity of the fse4 mutant were lower than WT. The endosperm of the mutant had many single dispersed starch granules with large spaces between each other. Using 1568 recessive individuals, FSE4 was narrowed down to a 252 kb region. Sequencing revealed a single base substitution in the first exon of the delta 1-pyrroline-5-carboxylate synthetase (P5CS), resulting in a conserved amino acid variation. Most of the genes related to starch synthesis were downregulated in fse4 and the protein accumulation related to starch synthase were reduced. The contents of various amino acids in fse4 rice flour were increased or decreased, the total free amino acids contents in fse4 seeds was 2.6 times higher than those in WT. Exogenous proline was applied during the germination of fse4 seeds, and the embryonic lethal phenotype was partially recovered.【Conclusion】FSE4 encode the key rate-limiting enzyme P5CS of proline synthesis, which plays an important role in the biosynthesis and metabolism of amino acids in endosperm and affects the accumulation of starch.     

Antimicrobial effects of black rice extract on Helicobacter pylori infection in Mongolian gerbil

It has been reported that cyanidin 3-O-glucoside (C3G) is an inhibitor of Helicobacter pylori toxin secretion. C3G is classified as an anthocyanin and is a major component of black rice extract (BRE). The present study aimed to identify a new functional food material to prevent H. pylori infection in Mongolian gerbil model. Toxicity in the liver and kidney were not detected after BRE administration (10 or 50 mg/kg). BRE treatment reduced bacterial colonization in animal gastric tissue, as well as infection signs as observed on the analysis of the hematological data. It was also found that the relative mRNA levels of the inflammatory cytokines were reduced in BRE-treated groups. These findings suggest that BRE acts as a potent inhibitor of H. pylori infection and pathogenesis in Mongolian gerbils. We propose that BRE may be used to manage gastroduodenal diseases caused by H. pylori infection.     

液化乙烷船液罐装载极限的提高方法

为了优化液化乙烷船远洋运输能力,根据液化气船液罐装载极限的基本要求和液化乙烷的货品属性,从提高基准温度下货物的对应密度和充装极限两个方面研究了提高液化乙烷船液罐装载极限的方法。将理论计算与液化乙烷船实际数据相结合,分析了BOG的不同回收利用方式对控制乙烷密度的影响;为了使主罐体充装极限达到99.5%,在液罐顶部设置气室,并将液罐压力释放阀安装于气室顶部;同时,对液位测量、温度测量、再液化设备启动压力设定、应急切断响应周期、液罐尺寸等修正裕量系数开展了研究,得出了在满足约束条件下提高装载极限的方法。结果表明:该方法可使得液化乙烷在基准温度下的对应密度、充装极限分别提高6.67%～8.89%、1.5%,可在其他液化乙烷船储运中推广应用。     

马尾松幼苗光合产物的运输与分配特征

【目的】解析光合产物在马尾松幼苗植株中的运输和分配规律,揭示马尾松生产力形成过程,为探讨不同环境干扰下马尾松光合产物分配过程的变化提供参考依据。【方法】采用 13 C同位素脉冲标记法,对1.5年生马尾松幼苗进行标记,标记结束后第0、2、5、17、24、72、120、168、216和 360 h ,按不同部位对马尾松幼苗进行全收获取样,测定 13 C含量,以监测近期合成的光合产物在马尾松幼苗中的运输和分配规律,同时测定光合产物全碳、非结构性碳水化合物(NSC)在各个器官的积累量。【结果】1)标记的光合产物在针叶中合成后,向各库器官的运输量随着时间延长由多逐渐减少,具体表现为标记结束后0~24 h内最多,24~216 h逐渐减少,216 h之后运输完成,且59%以上标记的光合产物在0~24 h内运输到各库器官。2)光合产物运输趋于稳定后,在各器官的分配大小依次为1年生叶>当年生叶>根>茎干>1年生枝>当年生枝,与生物量的分配大小一致,但与库活力大小不同,其库活力依次为当年生叶>当年生枝>1年生叶>根>1年生枝>茎干。3)各器官全碳和NSC积累量的分配与近期合成光合产物的分配大小一致,依次为1年生叶>当年生叶>根>茎干>1年生枝>当年生枝。【结论】马尾松幼苗光合产物运输速率大于0.1 m ·h -1 ,59%以上标记的光合产物在合成后的0~ 24 h 内完成向各个库器官输出。新合成的光合产物在各器官中的积累量表现为功能器官(叶和根)居多,这一分配规律有利于马尾松幼苗阶段的生长。     

基于不同林分类型下土壤碳氮储量垂直分布

以辽东大伙房水库周边防护林典型林分针阔混交林(落叶松-油松-刺槐混交林)、油松林、落叶松林、刺槐林为研究对象,对其土壤养分含量进行测定,研究了不同林分土壤剖面上有机碳、全氮、有机碳储量的分布规律。结果表明:随着土层深度的增大,4种林分的土壤有机碳、全氮含量均逐渐降低;4种林分土壤剖面有机碳含量大小顺序为落叶松林(24.16g/kg)刺槐林(23.07g/kg)针阔混交林(16.06g/kg)油松林(15.76g/kg);全氮含量大小顺序为刺槐林(5.23g/kg)落叶松林(4.57g/kg)油松林(3.45g/kg)针阔混交林(2.42g/kg);C/N平均值大小顺序为落叶松林(7.36)针阔混交林(6.51)油松林(4.67)刺槐林(4.57);4个林分0-40cm土层的有机碳储量大小为落叶松林(112.94t/hm~2)刺槐林(107.40t/hm~2)针阔混交林(105.42t/hm~2)油松林(89.89t/hm~2);4种林分土壤pH无明显差别,各土层土壤pH随土层深度增加而增大;4种林分土壤容重由高到低顺序依次为针阔混交林(1.73g/cm~3)油松(1.65g/cm~3)落叶松(1.64g/cm~3)刺槐(1.56g/cm~3)。4个林分土壤有机碳含量与土壤全氮含量互相间均存在极显著正相关关系,土壤有机碳、全氮含量与C/N之间则没有明显相关关系;在针阔混交林中,土壤容重、土壤全氮含量和土壤pH与土壤有机碳之间存在线性数量关系,而其他纯林则没有这种关系。