水稻白条纹叶突变体st11的遗传分析与基因定位

白条纹叶突变体st11是从粳稻品种Kitaake组培过程中获得的。该突变体在分蘖前叶色表现为正常,从分蘖期开始新生叶表现为白条纹直至成熟期。与野生型相比,该突变体的分蘖、株高、结实率和千粒重等农艺性状没有发生明显变化。遗传分析表明该突变体白条纹叶性状受一对隐性核基因控制。利用该突变体分别与水稻02428、Jodan杂交构建了两个F2群体用于基因定位。通过集群分离分析(bulked segregant analysis)发现该基因位于第1染色体端粒附近,并与分子标记RM151和RM10080连锁。进一步利用更多分子标记分析F2群体,我们将该基因定位于I10和I26两个标记之间大约270kb的区间内。     

小麦抗源南农790成株期抗条锈性遗传分析与分子作图

为明确小麦抗锈种质南农790条锈病抗性特征和抗病遗传规律,以5个条锈菌生理小种CYR23、CYR29、CYR31、CYR32和CYR33对其进行苗期抗谱鉴定,并以CYR32和CYR33混合小种对南农790、Avocet S及南农790与Avocet S正交所获得的F1代、F2代和F2∶3群体进行混合小种成株期抗条锈性鉴定及遗传分析。抗病鉴定结果显示,南农790苗期对CYR32和CYR33表现为中度感病(IT:6~7),对其余小种表现为抗病(IT≤2),成株期对混合小种表现为近免疫(IT≤2,S≤5%),表明南农790具有成株期抗条锈性;各世代抗病遗传分析结果表明,其成株期抗病性由一个显性单基因控制,暂命名为YrNan;采用集群分离分析法(BSA),以SSR分子标记对F2代分离群体进行分子作图,发现了6个与YrNan连锁的SSR标记(Xgwm11,Xbarc187,Xbarc181,Xgwm273,Xwmc419和Xwmc216),并将其定位于小麦的1BL染色体,两侧最近的标记分别为Xbarc181(1.3cM)和Xgwm273(6.3cM)。研究结果为南农790抗条锈病基因的分子标记辅助选择及在育种上的应用提供了依据。     

抗逆相关基因GmDREB导入农杆菌的研究

利用改进的冻融法和电击法成功地将带有编码DREB转录因子GmDREB基因的prdGm-200双元载体分别导入根癌农杆菌EHA101、EHA105和LBA4404菌株中,同时探讨了影响冻融法和电击法转化效率的因素。结果表明:农杆菌细胞OD600约为0.6时冻融法和电击法的转化效率均较高。在利用冻融法转化农杆菌时,新制备的感受态细胞有利于提高转化效率。在进行电击法转化农杆菌时,电场强度和脉冲时间都对农杆菌的转化效率有影响。     

流产布鲁氏菌BCSP31基因在大肠杆菌中的可溶性高效表达

设计1对引物,突变除去布鲁氏菌BCSP31(细胞表面蛋白31)基因的N端信号肽氨基酸序列,经NotⅠ和EcoRⅠ酶切后与相同处理的表达载体pGEX-4T-1连接,转化BL21(DE3)感受态细胞。酶切及PCR扩增鉴定,获得阳性克隆,测序证明其阅读框完全正确。用IPTG诱导表达构建成功的阳性克隆,对产物进行SDS-PAGE和Western-blotting检测,发现表达产物出现预期分子量57 kD的融合蛋白,该蛋白能与牛流产布鲁氏菌阳性血清发生反应,进一步证明目的蛋白主要以可溶性形式存在,表达量约占总蛋白的40%,应用GST琼脂糖凝胶FF纯化可得到纯度较高的表达产物。     

Dynamics of nitrogen translocation from mature leaves to new shoots and related gene expression during spring shoots development in tea plants (Camellia sinensis L.)

The contribution of N remobilization is crucial for new shoots growth and quality formation during spring tea shoots development. However, the translocation mechanism of N from source leaves to sink young shoots is not well understood. In the present study, ¹⁵N urea was applied to mature tea leaves one week before bud break to track N remobilization in a field experiment. The dynamic changes in plant ¹⁵N abundance, contents of amino acids, and the expression levels of genes related to N metabolism and translocation were followed during the 18‐d development of new spring shoots until three expanding young leaves. The results showed that during the growth of new shoots the amount of ¹⁵N in the shoots increased, whereas the N_dff (N derived from ¹⁵N‐urea) in mature leaves decreased, showing that the foliar‐applied N in mature leaves was readily exported to new shoots. This process was found to be accompanied by decline of chlorophylls. In the mature leaves, expression CsATG18a and CsSAG12 involved in autophagy was dramatically induced (> 4‐fold) at approximately nine days after the bud breaking. The genes involved in the transformation of amino acids, including primarily CsGDH2, CsGDH4, CsGLT3, CsGS1;3, and CsASN2 were upregulated by > 3‐fold after bud breaking. The expression levels of CsATG8A, CsATG9, CsSAG12, CsGS1;1, CsGDH1, and CsAAP6 correlated negatively with the N_dff in mature leaves, but positively with ¹⁵N amount and total N amount in new shoots, suggesting these genes played important roles in N export from mature leaves. In the new shoots, the expression of most genes showed two defined peaks, one on six days and one on 12 days after bud breaking. The expression of CsGS2, CsASN3, CsGLT1, and CsAAP4 positively correlated with the ¹⁵N amount and total N amount in new shoots. These genes might be involved in the transport and re‐assimilation of N from mature leaves. The overall results demonstrated that the translocation of ¹⁵N from mature leaves to new spring shoots was regulated by the genes involved in autophagy, protein degradation, amino acid transformation and transport.     

施氮量和采收时期对油蔬两用型油菜菜薹维生素合成的影响

为明确施氮量和采收时期对油蔬两用型油菜薹维生素含量的影响,以富硒1号为材料,采用裂区试验设计,设置3个施氮量(120、180和240 kg·hm^-2)和4个采收时期(以株高20、30、40和50 cm表示),分析了油菜薹维生素C、维生素E、维生素B₁和维生素B₆含量的变化;比较了半乳糖内酯脱氢酶(GalLDH)、抗坏血酸氧化酶(AAO)、抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)和单脱氢抗坏血酸还原酶(MDHAR)活性,并分析了维生素家族基因在菜薹中的表达量。结果表明,随着施氮量的增加,油菜薹还原型维生素C(AsA)含量降低;氧化型维生素C(DHA)含量在株高20 cm采收时增加,但在30 cm及以上株高采收时则降低;维生素E和维生素B₆含量先增加后降低;维生素B₁含量增加;维生素C合成关键酶GalLDH活性先略增加后降低;AAO和DHAR活性增加,APX和MDHAR活性则整体表现为先增加后降低。且施氮量增加后,维生素家族各基因在株高30和40 cm采收的菜薹中主要表现为正向调控,而在株高20和50 cm采收的菜薹中主要表现为负向调控。就采收时期而言,菜薹中的维生素含量在株高30~40 cm时高于其他时期;在同一施氮量下,随着菜薹采收株高的增加,维生素家族基因的表达量主要呈上调模式。综合分析可知,GalLDH活性的降低以及AAO/DHAR和APX/MDHAR代谢平衡是增施氮肥导致菜薹维生素C含量降低的重要原因。本研究结果为油蔬两用型油菜薹优质栽培技术提供了理论依据。     

Toxicity of cadmium and its competition with mineral nutrients for uptake by plants: A review

Cadmium(Cd)is a toxic heavy metal occurring in the environment naturally and is also generated through various anthropogenic sources and acts as a pollutant.Human health is affected by Cd pollution in farmland soils because food is the main source of Cd intake in the non-smoking population.For crops,Cd toxicity may result from a disturbance in uptake and translocation of mineral nutrients and disturbance in plant metabolism,inhibiting plant growth and development.However,plants have Cd tolerance mechanisms,including restricted Cd uptake,decreased Cd root-to-shoot translocation,enhanced antioxidant enzyme activities,and increased production of phytochelatins.Furthermore,optimal supply of mineral nutrients is one of the strategies to alleviate the damaging effects of Cd on plants and to avoid its entry into the food chain.The emerging molecular knowledge contributes to understanding Cd uptake,translocation,and remobilization in plants.In this review,Cd toxicity and tolerance mechanisms,agricultural practices to minimize Cd accumulation,Cd competition with essential elements(calcium,copper,iron,zinc,and manganese),and genes associated with Cd uptake are discussed in detail,especially regarding how these mineral nutrients and genes play a role in decreasing Cd uptake and accumulation in crop plants.     

芹菜水孔蛋白基因AgPIP2;1的克隆及其对非生物胁迫的响应

为了研究芹菜质膜内在蛋白基因的序列特征及其在非生物胁迫条件下的表达特性,探讨其抗逆功能,以芹菜品种六合黄心芹为试验材料,通过RT-PCR技术克隆AgPIP2;1基因,通过生物信息学软件分析其核苷酸和氨基酸序列特征,并采用荧光定量PCR技术检测其在不同组织的表达及其对非生物胁迫的响应。结果表明,AgPIP2;1基因含有1个861 bp的开放阅读框,编码286个氨基酸,其编码的蛋白属于PIP2类蛋白。氨基酸序列分析显示,AgPIP2;1含有高度保守的NPA基序以及高等植物PIP蛋白的保守序列,与其他物种PIP2类蛋白具有较高的同源性,与胡萝卜DcPIP2;1的氨基酸序列同源性达到97.90%。在亲缘关系上与大豆GmPIP2;1、胡萝卜DcPIP2;1和绿豆VrPIP2;1较为接近。实时荧光定量PCR技术分析表明,AgPIP2;1基因在芹菜根、叶柄和叶片中均有表达,其中在叶片中的表达量最高,在根中的表达量最低,呈现比较明显的组织特异性。此外,AgPIP2;1基因受到高温、低温、干旱和盐等非生物胁迫的诱导,说明该基因可能参与芹菜抵御非生物胁迫的过程。本研究为进一步了解AgPIP2;1基因的功能及其在非生物胁迫中的作用奠定了基础。     

大白菜抽薹相关基因BrFLC1的KASP标记开发

为了提高大白菜耐抽薹品种的分子育种效率,本研究针对大白菜抽薹相关基因BrFLC1第6个内含子的第一个碱基(Pi6+1)G-A的SNP变异开发进行高通量检测的竞争性等位基因特异性PCR(KASP)标记。结果表明,开发的KASP标记BrFLC1-KASP1可有效将晚抽薹类型材料Y177-12(G)和早抽薹类型材料Y195-93(A)分为2组。利用BrFLC1-KASP1标记可以对57份大白菜材料的基因型进行有效鉴别,且鉴定结果与酶切扩增多态性标记(CAPS)G-MvaI以及直接测序法的鉴定结果完全一致。综上所述, BrFLC1-KASP1标记在大白菜材料中具有通用性,且具有准确率高、成本低、效率高的特点。本研究结果对大白菜耐抽薹性的分子标记辅助选择具有重要的育种实践价值。     

茶树CsSNAT基因的克隆与表达分析

为明确茶树SNAT基因的序列特征和表达特点,在茶树转录组测序的基础上以茶树(Camellia sinensis)品种舒茶早为试验材料,通过SMART^TM RACE技术克隆茶树褪黑素合成途径中关键限速酶5-羟色胺-N-乙酰转移酶CsSNAT基因的cDNA全长序列,并分析其基因结构和表达模式。结果表明,CsSNAT基因序列全长1 014 bp,其中包含742 bp的完整开放阅读框(ORF),编码247个氨基酸,预测等电点7.64,蛋白分子量27.36 kDa。氨基酸序列比对显示,CsSNAT蛋白与其他高等植物的SNAT蛋白具有较高同源性,与葡萄 VvSNAT(CBI31163)同源性最高,为66.19%。诱导蛋白最佳条件为温度28℃,异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.5 mg·L^-1,诱导时间6 h,在上清液中可获得大量分子量为66 kDa的可溶性融合蛋白。实时定量 PCR 分析表明,CsSNAT在褪黑素处理6 h后表达量最高,为对照的3倍;茶尺蠖取食6 h后,CsSNAT表达显著上调。不同激素处理结果显示,CsSNAT受脱落酸(ABA)、茉莉酸甲酯(MeJA)和水杨酸(SA)诱导表达。本研究结果为5-羟色胺-N-乙酰转移酶功能研究奠定了一定的基础。