利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

Integration and expression of Sorghum C4 phosphoenolpyruvate carboxylase and chloroplastic NADP-malate dehydrogenase separately or together in C3 potato plants

We have integrated two cDNAs expressing Sorghum photosynthetic phosphoenolpyruvate carboxylase (C₄-PEPC) and NADP-malate dehydrogenase (cpMDH), two key enzymes involved in the primary carbon fixation pathway of NADP-malic enzyme-type C₄ plants, separately or together into a C₃ plant (potato). Analysis of the transgenic plants showed a 1.5-fold increase in PEPC and cpMDH activities compared to untransformed plants. Immunolocalization confirmed an increase at the protein level of these two enzymes in the transgenic plants and indicated that the Sorghum cpMDH was specifically addressed to the chloroplasts of potato mesophyll cells. However, integration of either or both of the cDNAs into the potato genome did not appear to significantly modify either tuber starch grain content or the rate of photosynthetic O₂ production compared to control untransformed plants. The low level of transgene expression probably explains the lack of influence on carbon metabolism and photosynthetic rates. This general observation suggests that some complex mechanism may regulate the level of production of foreign C₄ metabolism enzymes in C₃ plants.     

不同光合类型牧草对干旱-复水的光合生理响应及生长适应策略

基于干旱频率增加、强度增大这一全球降水变化背景, 探究干旱-复水条件下不同功能群(C₃和C₄)植物的光合生理响应及生长适应策略有助于预测降水格局变化条件下草地的植被组成和生态系统功能。该研究采用盆栽实验, 以松嫩草地生长的一年生C₃ (4种)和C₄ (3种)牧草为实验材料, 设置了对照、中度干旱和重度干旱3个水分处理水平, 在干旱末期及复水期对植物进行气体交换、生物量和比叶质量的测量。在干旱条件下, 各物种净光合速率和气孔导度均呈下降趋势, 水分利用效率呈上升趋势。干旱对不同植物光合指标的影响存在功能群差异, 随干旱程度的增加C₄植物逐渐丧失光合优势, 重度干旱对C₄植物净光合速率的影响较C₃植物更加明显。由于干旱条件下C₃植物光合固碳主要受气孔限制而C₄植物主要受代谢限制, 因此复水后C₄植物净光合速率恢复速度较C₃植物慢。干旱条件下, 各物种的生物量降低, 根冠比和比叶质量升高, 干旱对C₃植物各生长指标的影响均大于C₄植物; 复水处理后, C₃植物生物量随干旱强度增加呈下降趋势, 而C₄植物的生物量与对照相比无显著差异。     

利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

AtERF49在拟南芥应答盐碱胁迫中的作用

盐碱胁迫是造成作物减产的主要逆境因素之一。植物AP2/ERF（APELATA2/ethylene response factors）转录因子在植物生长发育及其响应非生物逆境胁迫过程中发挥重要作用。探究AtERF49在拟南芥中对盐碱胁迫的应答,为深入解析AtERF49参与植物对盐碱胁迫的分子机理奠定基础。选取拟南芥野生型Col-0、过表达AtERF49转基因拟南芥和CRISPR/Cas9突变体erf49为试验材料,用150 mmol/L混合盐碱（摩尔比NaHCO₃∶Na₂CO₃=9∶1）溶液进行处理,使用荧光定量PCR技术对该基因的基本特性、盐碱胁迫及光合响应基因表达模式等进行分析。结果表明,盐碱胁迫处理后,突变体erf49叶片萎蔫并发生白化,而过表达AtERF49植株叶片稍有变黄。此外,在盐碱胁迫条件下,过量表达AtERF49上调盐碱胁迫响应基因（RD29A和RAB18）以及光合响应基因rbcL的表达。拟南芥叶片叶绿素荧光参数测定结果表明,过表达AtERF49植株的光系统Ⅱ实际量子产能Y（Ⅱ）、光化学淬灭系数（qP）显著高于Col-0,光损伤程度（NO）和非光化学淬灭系数（qN）显著低于Col-0,而突变体erf49与之相反。因此,AtERF49通过调控下游盐碱胁迫响应基因的表达以及植物的光合作用效率,改变参与植物对盐碱胁迫的应答。     

Modification of nitrogen remobilization, grain fill and leaf senescence in maize (Zea mays) by transposon insertional mutagenesis in a protease gene

A maize (Zea mays) senescence-associated legumain gene, See2beta, was characterized at the physiological and molecular levels to determine its role in senescence and resource allocation. A reverse-genetics screen of a maize Mutator (Mu) population identified a Mu insertion in See2beta. Maize plants homozygous for the insertion were produced. These See2 mutant and sibling wild-type plants were grown under high or low quantities of nitrogen (N). The early development of both genotypes was similar; however, tassel tip and collar emergence occurred earlier in the mutant. Senescence of the mutant leaves followed a similar pattern to that of wild-type leaves, but at later sampling points mutant plants contained more chlorophyll than wild-type plants and showed a small extension in photosynthetic activity. Total plant weight was higher in the wild-type than in the mutant, and there was a genotype x N interaction. Mutant plants under low N maintained cob weight, in contrast to wild-type plants under the same treatment. It is concluded, on the basis of transposon mutagenesis, that See2beta has an important role in N-use and resource allocation under N-limited conditions, and a minor but significant function in the later stages of senescence.