B3基因家族是植物中特有的转录因子家族,在植物生长发育及胁迫响应中有重要作用.为探究B3基因家族在玉米(Zea mays)中的基本信息及生长发育和逆境胁迫中的生物学功能,本研究对玉米全基因组B3家族成员进行鉴定并分析相关信息,依据转录组数据分析B3基因家族组织特异性表达及胁迫处理下的表达响应.结果显示,玉米B3基因家族包含88个成员,分为ABI、ARF、HSI、RAV和REM 5个亚家族,均含有B3结构域,同一个亚族成员的基因结构及保守基序(数量、类型)相似.分析发现,B3成员基因的启动子区域中均富含激素及逆境响应等功能元件.组织特异性表达分析结果显示,大部分B3基因在分生组织中优势表达.对B3家族成员在冷、热、盐、紫外和干旱这5种胁迫因子的响应表达分析显示,5个亚家族成员均可不同程度参与响应冷、热、盐、紫外和干旱胁迫因子,其中HSI亚家族中的所有成员均参与了对紫外胁迫因子的响应途径.以上结果阐明了B3基因家族在玉米全基因组中的分布、结构和系统发育特征,并依据玉米转录组数据,初步推测其在生长发育和胁迫应激响应中发挥重要作用,为深入研究B3基因家族在玉米中的功能与分子机制提供了数据基础.

为探究玉米生物钟基因ZmPRR1-2的功能及表达特性,解析玉米光周期途径调控开花的机理,该研究以玉米骨干自交系'黄早4'为材料,克隆ZmPRR1-2基因的cDNA序列并进行生物信息学分析;利用qRT-PCR技术对该基因进行组织特异性表达分析和48 h的昼夜节律表达分析.结果表明:(1)成功克隆获得ZmPRR1-2基因的编码区全长1554 bp,编码517个氨基酸,编码的蛋白属于PRR基因家族,含有1个REC结构域和1个CCT结构域,多序列比对和系统进化分析显示ZmPRR1-2基因在禾本科植物中高度保守;ZmPRR1-2蛋白属亲水性蛋白,不包含跨膜结构域和信号肽.(2)ZmPRR1-2基因在玉米叶片中的表达量最高,显著高于其他7个组织,表明该基因主要在叶片中发挥功能,而在果穗和花丝中表达量相对较低,且显著低于雄穗中的表达量.(3)昼夜节律表达分析显示,在短日照条件下,ZmPRR1-2基因的表达量于光照3 h后开始逐渐上升,在光照结束后3 h时达到表达高峰;在长日照条件下,于光照6 h后ZmPRR1-2基因的表达量才开始逐渐上升,且在光照结束时达到表达高峰.研究认为,ZmPRR1-2基因在玉米开花转化过程中对雄穗发育和雌穗发育的调控功能存在差异,推测Zm-PRR1-2基因可能在玉米生物钟调控中发挥重要作用.

克隆玉米伪应答调控基因ZmPRR1-1启动子并对其序列进行分析,以期为深入研究ZmPRR1-1基因的调控机制及基因功能提供参考.以玉米黄早4的叶片为供试材料,参考已公布玉米自交系CML247基因组中Zm-PRR1-1启动子的序列信息设计引物,克隆ZmPRR1-1基因的启动子,并利用生物信息学对启动子中的核心区域、顺式作用元件、转录因子结合位点和CpG岛进行预测分析.克隆得到长度约2600 bp的ZmPRR1-1启动子,预测ZmPRR1-1启动子可能存在2个核心启动子区域,除了存在核心元件TATA-box和启动子增强元件CAAT-box外,还存在光响应、激素应答、胁迫响应等顺式作用元件.转录因子结合位点分析表明,ZmPRR1-1启动子区中预测包含ERF在内的6种转录因子家族,此外在启动子区域中预测存在4个符合限定条件的CpG岛区域.玉米ZmPRR1-1启动子区域存在丰富的顺式作用元件和转录因子结合位点,暗示ZmPRR1-1可能受到外界多种类型调控因子的调控并启动转录起始,且在多种调控网络中发挥功能.

借助生物信息学手段,从玉米DREB家族基因的鉴定、基因结构、顺式作用元件等多角度进行解析,以期了解其在抗逆途径中的潜在功能.结果表明:玉米中共有87个DREB家族成员,分为6个亚组,理化性质分析结果表明其编码的氨基酸序列长度为125～452个氨基酸,均编码亲水性蛋白;在玉米10条染色体上均有分布,在4号、5号染色体长臂处较为集中;仅有少数基因含有内含子,大多数成员只以CDS的形式存在且在各亚组之间保守;启动子区域除含有MBS等响应干旱胁迫的元件外,还含有响应赤霉素、ABA、MeJA等相关调控途径的元件;物种共线性分析结果表明,高粱与玉米DREB家族成员之间存在共线性特征,且部分基因呈现倍性关系;基于转录组数据的表达模式分析显示,在干旱、盐、干旱和盐双重胁迫处理下,仅有几个基因的表达水平显著升高,多数DREB家族基因以下调的表达方式参与胁迫响应.

[目的]FKF1是多种植物开花途径中发挥重要作用的关键基因.为研究玉米FKF1功能,利用CRISPR/Cas9基因编辑技术将ZmFKF1进行定点编辑,获得ZmFKF1编辑突变体.同时以此为材料,通过表型分析及关键开花基因的表达量变化分析,明确ZmFKF1在玉米开花途径中的作用,为玉米分子育种及遗传改良提供理论依据.[方法]以玉米B104为材料,克隆ZmFKF1,通过序列比对明确ZmFKF1的基因结构.以ZmFKF1为靶标基因,根据CRISPR/Cas9技术原理设计靶点,将设计的靶点序列在玉米参考基因组中进行比对分析,排除非特异性靶位点,最终筛选出在ZmFKF1第1外显子上的靶位点ZmFKF1-T1,构建CRISPR/Cas9基因编辑表达载体.利用农杆菌介导法,转化玉米B104幼胚,通过抗性筛选获得抗性愈伤组织,之后诱导出芽和生根,获得T0代ZmFKF1编辑阳性植株,并利用cas9特异引物进行验证.利用靶位点扩增测序法,明确T1代ZmFKF1编辑植株在ZmFKF1预期靶标位点是否发生突变及突变的类型,筛选获得ZmFKF1定点突变纯合株系.获得这些材料后,以野生型B104为对照,统计和分析开花表型.同时,利用实时荧光定量PCR技术检测上述材料中与玉米开花途径相关的关键基因的表达量变化,对表型进行进一步的验证.[结果]在玉米FKF1的第1外显子上设计靶点构建基因编辑表达载体,通过遗传转化获得的转基因株系实现了对ZmFKF1的定点突变,共获得T0代ZmFKF1编辑阳性株系18株,在预期靶标位点上发生突变的有6株,2种不同的突变类型:单碱基插入和多碱基缺失.通过开花时间统计和分析,与野生型B104相较,3个T2代ZmFKF1编辑纯合突变体的开花时间延迟,显著(P<0.05)晚于B104.进一步对这些材料中的开花关键基因ZmGI、conz1和ZmZCN8进行表达量检测,发现突变体中这些基因的表达明显(P<0.05)低于野生型B104,与晚花表型相符.[结论]可利用CRISPR-Cas9技术对ZmFKF1进行定点编辑获得基因编辑突变体,且突变体开花时间明显延迟.

[目的] 分析ZmCOL3基因编码蛋白的结构及功能,预测ZmCOL3基因可能具备的功能,为该基因在玉米开花调控及其他功能的研究提供思路.[方法] 通过生物信息学的方法对ZmCOL3基因编码蛋白质的理化性质、保守结构域、二级结构、三级结构、信号肽、跨膜结构域、亚细胞定位及启动子顺式作用元件进行了预测,并对该基因在玉米不同组织的表达量进行了分析.[结果] ZmCOL3基因所编码的蛋白包含335个氨基酸,相对分子质量为35.39 kD,理论等电点为5.04,属于酸性蛋白,具有亲水性和不稳定性,含有1个CCT结构域和1个B-box结构域,没有信号肽和跨膜结构域,主要定位在细胞核中,其二级结构主要是由无规则卷曲组成,其次是α螺旋,还含有少量的延伸链和β转角,同源建模相似度为64.15％,该基因的启动子不仅含有TATA-box、CAAT-box启动子基本的顺式作用元件,还含有Sp1等光响应作用元件,以及脱落酸、茉莉酸甲酯等激素响应元件.在玉米的种子、初生根、节间、叶、雌穗和雄穗等6个部位中,叶片中ZmCOL3基因的表达量最高,其次是雄穗,在种子中该基因表达量最低,总体上看ZmCOL3基因在叶片中的表达量显著高于其他部位.[结论] ZmCOL3蛋白是一个亲水、不稳定的酸性蛋白,其含有CCT结构域和B-box结构域,符合CCT基因家族中COL亚家族的结构特征,属于该家族成员,可能参与生物钟的调控来影响玉米开花.启动子顺式作用元件分析发现ZmCOL3基因的启动子包含光响应及各种激素响应元件,推断该基因可能受到光周期调控和激素等非生物胁迫调控,暗示其可在多重反应调控网络中发挥作用.

为研究CDF1在玉米光周期开花途径中的作用,本文确定了ZmCDF1的亚细胞定位及其与ZmGI的互作关系.本文以玉米自交系B73为材料,克隆获得了玉米ZmCDF1的全长编码序列(coding sequence,CDS),并与其他6个物种进行了氨基酸序列比对.结果 显示ZmCDF1基因含有2个外显子,3个内含子,CDS序列全长1 296 bp,编码432个氨基酸,包含一个典型的Dof (DNA binding with one finger)结构域.亚细胞定位显示,ZmCDFI定位于细胞核中.酵母双杂交和荧光素酶互补实验显示ZmCDF1能够与玉米光周期途径的重要因子ZmGI互作.上述结果暗示ZmCDF1在玉米光周期途径中发挥重要作用.本研究为玉米光周期开花途径提供了新的基因资源.

有效地利用耐密性品种,通过合理增加种植密度来提高单产是玉米取得高产的主要途径.以4个玉米品种为材料,在5个种植密度下分析产量及其构成要素对种植密度的响应.结果表明,随着种植密度的提高,群体产量呈先增加后降低的变化趋势,科大216的耐密性最好,其适宜种植密度为9.0万株/hm2.

《试验设计与数据分析》是农学、生物科学和林学等专业重要的基础课.在高通量数据时代,利用网络资源,提升教师自身知识水平;在优化理论课程内容、改革教学方式和手段、利用现代软件等方面不断完善教学体系建设,培养学生统计思维和实践能力,提高该课程的教学质量和教学效果.

开花是植物由营养生长向生殖生长转变的重要过程,光周期是调控这一过程的关键环境因素之一.在高等植物开花的光周期途径中,生物钟处于关键位置,在调控植物开花中发挥重要作用,它能对输入信号进行振荡处理,并传递给下游基因,同时保持自身节律性,从而调控开花.伪应答调控蛋白(PRRs)作为生物钟中央振荡器的重要组成部分,主要作用是调控下游基因的表达,参与昼夜节律的调节.本文综述了PRRs家族的结构特征、表达规律、成员之间的调控关系及其在光周期调控网络中的作用,为进一步研究PRRs基因家族的分子功能及互作提供了一定的理论参考.

Photoperiodic pathway is one of the important ways to regulate the flowering time in plants,during the long evolutionary process,plants have formed an extremely precise and sophisticated light-sensitive system in the photoperiod pathway.Plants receive the light signal which is mainly completed by the photoreceptor of light receptor system.The ZTLs family is a class of blue light receptor proteins which regulate the degradation of proteins in plant,ubiquitination enzymes,which play an important role in the light signal entering the circadian clock and regulating the expression of flowering genes downstream.In this study,reviewed and predicted the research on the structure and function of ZTLs family proteins in different plants,and provides a certain theoretical basis for understanding and studying the mechanism of ZTLs family in regulating flowering through photoperiodic pathway.

To investigate the functions of LRR-RLKs gene RLK6 in Arabidopsis,a pSuper1300-RLK6 expression vector was constructed and five RLK6 overexpression transgenic lines were obtained.The two lines,OE8 and OE9,were selected for further analysis.The homozygous RLK6 mutants (rlk6-2,rlk6-3 and rlk6-6),OE8,OE9 and WT were characterized for phenotype analysis,and the result showed that RLK6 mutants appeared to show similar phenotype with wild-type (WT) during growth and development.But,OE lines displayed obviously early flowering than WT.Four key genes which relates to flowering were examined in all genotypes by using qRT-PCR.In OE lines,increased expression of floral activator genes (CO,SOC1 and LFY) with the decreased level of floral repressor gene (FLC) was observed,which was consistent with it phenotypes,when compared to WT.These results suggest that RLK6 play a key role in promoting flowering in Arabidopsis,and these results will lay the foundation for understanding the mechanisms of RLK6.

实践教学体系是由实践教学活动各个要素构成的有机联系的总体,包含基本素质、基础技能、专业技能和综合训练4个层次以及不同层次下的素质拓展、公共基础技能、农学基础技能、种子专业技能、种子专业综合能力、综合实习、科研训练、科技创新等8个模块.在不同模块下开设的实验(实践)课440学时,14学分,实践教学周数46周,46学分,加上其他实践教学,实践学分占185总学分的三分之一.

In eukaryotic cells, the exosome is an evolutionarily conserved macromolecular complex with nine subunits, mediating numerous reactions of 3'→5' RNA processing and degradation. To get around the interac-tion relationship between the RRP41-LIKE (RRP41L) and other subunits of exosome in Arabidopsis thaliana, yeast two-hybrid assay and ifrelfy luciferase complementation imaging assay (LCI) were used to conifrm the in-teraction relationship between RRP41L and other two core subunits of exosome, RRP42 and RRP45B in vitro and in vivo. Our results showed that RRP41L interacted with the RRP45B, but not interacted with the RRP42.

为提高学生素质，对种子科学与工程专业实践教学体系进行了探索和实践。实践教学体系是由实践教学活动各个要素构成的有机联系的总体，包含基本素质、基础技能、专业技能和综合训练4个层次以及不同层次下的素质拓展、公共基础技能、农学基础技能、种子专业技能、种子专业综合能力、综合实习、科研训练、科技创新等8个模块。构建了“四层次八模块的实践教学体系”。在不同模块下开设的实验（实践）课440学时，14学分，实践（实习）教学周数46周，46学分，以及在其他方面未计算在内的实践教学环节和学分，实践教学总学分达60学分以上。经过4年一个教学周期的实践，按此实践教学体系运行，教学秩序正常，学生考研、就业情况良好，达到预期效果。

种子科学与工程专业要求的最低学分为185学分，课程模块设置为通识教育课程、农学学科平台课程、种子科学与工程专业方向课程、实践教学等4个模块，课内总学时为2808学时，4年教学总周数为162.5周。

High yield cultivation under field conditions,the effects of different agents,corn hybrids enzyme activity in rhizosphere soil after KD16 different concentrations. The results showed that: The rhizosphere enzyme-rhizosphere urease,alkaline phosphatase,protease,invertase and catalase activity of KD16 showed a trend of decreasing after increasing under different CT and CIT concentration treatments. However rhizosphere soil dehydrogenase activity showed a trend of declining. Compared with the CK,different concentrations of seed-coating agent CT and CIT had the most significant effect on rhizosphere urease,alkaline phosphatase,protease,invertase,catalase and dehydrogenase activity at seedling stage,and there was a positive correlation between differentiation and seed-coating agent concentration. In jointing stage,huge bellbottom stage and flowering stage,smaller CT and CIT concentration treatments had a greater influence on rhizosphere enzyme compared with seedling stage. The different treatments of CT and CIT significantly reduced the infection rate of corn plant, maked the corn plant grow stronger, and then promoted three major determinants of corn yield,increased significantly compared with the CK. In the actual production,producer can achieve the goal of high yield by using seeds mixed with seed-coating agent,and the most appropriate concentration were CT2 and CIT2.

采用PCR技术克隆得到拟南芥类核糖体RNA加工蛋白41(Ribosomal RNA-processing protein 41-LIKE,RRP41L)基因,然后构建其原核表达载体,并对其进行诱导和纯化,以期获得较大量的重组蛋白.结果表明,试验获得了拟南芥RRP41L基因的全长编码序列(coding sequence,CDS) (771 bp),将其与原核表达载体PGEX4T-1连接构建了原核表达载体PGEX4T-RRP41L,将PGEX4T-RRP41L质粒导入大肠杆菌(E.coli)BI21(DE3)中,在37℃条件下,用1 mmol· L-1 IPTG诱导8h后体外纯化融合蛋白.SDS-PAGE电泳结果显示,在此诱导条件下,PGEX4T-RRP41L重组质粒可表达出较大量的重组蛋白,蛋白质相对分子质量大小为53.4 kD,除去PGEX4T-1自身表达相对分子质量大小为26.0 kD后,与RRP41L编码的蛋白质相对分子质量约为27.4 kD的大小一致.

Pot and field experiments combined with laboratory analysis were carried out to study the effect of different ratios of drug to seeds of CT and CIT such as 1 ∶40,1 ∶20,1 ∶30 respectively on the emergence,seedling traits and physiological indicators of hybrids maize named Ke da 16.Results showed that all the different types of seed-coating decreased the seed germination potential,germination rate,germination index,and the decreasing effect was more significant with greater concentration.The seedling growth of maize was significantly influenced,and the height,root length,fresh weight and leaf area increased significantly,and significant change was observed on dry weight between treatments and CK.Moreover,significant effect was observed on physiological indicators of seeds,the amount of chlorophyll,soluble protein,proline decreased significantly compared with CK,which was positively correlated with the drug concentration,but the amount of soluble sugar of all treatments increased significantly compared with CK.SOD,POD,CAT activity and MDA content in seedling were determined to assess the stress physiology.Results showed that different seed-coating drugs with different concentrations made POD,SOD,CAT activity increased,which was positively correlated with the drug concentration.And the seedling MDA increased significantly compared with CK,which also was correlated with the drug concentration.The treatment of different drug concentrations significantly prevented the rough dwarf diseases,the highest percentage of yield increase was CIT2 treatment(15.8%),while the lowest was CT1 stimulation(2.46%).

根据自交系的农艺性状进行聚类,将90个自交系聚为7个类群,即旅大红骨群、reid群、紫红群、5878群、白粒群、P 78599群和唐四平头群。其结果与已知的亲缘关系基本符合,并讨论了个别不一致的原因,聚类结果为今后玉米自交系选配组合提供参考。