A mutation confers Monochoria vaginalis resistance to sulfonylureas that target acetolactate synthase

Acetolactate synthase (ALS) is the target enzyme for four distinct families of compounds: sulfonylureas (SUs), imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We cloned and sequenced the fragments encoding ALS genes from biotypes of Monochoria vaginalis susceptible (S) and resistant (R) to SU-herbicides. The nucleotide sequences of the 39 bp Domain A region for R M. vaginalis biotype differed from that of the S biotype by a single nucleotide substitution at variable Pro codon of Domain A (CCT to TCT), predicting a Pro in the S but a Ser in the R biotype. No nucleotide differences between S and R M. vaginalis were observed in Domain D. We suggest that the amino acid substitution at Domain A region is responsible for resistance to SU-herbicides in M. vaginalis collected from Ushiku City, Ibaraki Prefecture, Japan.     

小麦淀粉颗粒蛋白-1突变体挖掘及其相关农艺性状和品质特点的分析

小麦淀粉颗粒蛋-1(starch granule protein 1,即SGP-1)突变体是选育高直链淀粉含量、高抗性淀粉小麦品种的重要基础材料。SGP-1突变缺失,使胚乳中支链淀粉侧链的延伸受阻,从而使胚乳表观直链淀粉含量相对升高,所制食品的抗性淀粉含量也相应提高,具有重要的保健意义。本研究主要是利用SDS-PAGE方法,检测307份国内外农家种、育成种、高代品系及国内外黑小麦材料的SGP-1(SGP-A1、SGP-B1和SGP-D1)蛋白组成,进而发掘SGP-1突变体;并对突变体材料进行相关农艺性状和品质性状特点的分析,为育种利用及品质改良提供参考。结果共检测到4份SGP-1突变体,均为冬性材料,其中国外材料3份(CH62444、EAP74961和Amigo)、高代品系1份(05黑初8),分别为SGP-A1单缺体,SGPA1和-D1双缺体,SGP-A1和-B1双缺体,以及SGP-A1单缺体。在主要农艺性状方面,EAP74961、05黑初8和Amigo抽穗期与石4185(CK)相近,而CH62444则抽穗较晚,并且3份国外材料植株均较高。这4份突变体材料麸皮中的非水溶性戊聚糖含量相对于水溶性戊聚糖占绝对优势,而胚乳中非水溶性戊聚糖含量与水溶性戊聚糖含量则较为接近,但CH62444、Amigo和05黑初8麸皮中的总戊聚糖含量较高(分别为14.33%,15.20%和12.91%),而EAP74961面粉中的总戊聚糖含量(1.34%)较高。在淀粉组分含量方面,4份突变体材料差异不大。     

小麦矮缩病毒引起的植株矮化与赤霉素代谢的相关性分析

【目的】由异沙叶蝉(Psammotettix alienus)传播的小麦矮缩病毒病是近年来中国西北部麦区严重发生的小麦病毒病害之一。受侵染的小麦植株严重矮化,有效分蘖减少,产量损失严重。论文旨在明确小麦矮缩病毒(Wheat dwarf virus,WDV)侵染小麦植株后矮化症状形成与赤霉素代谢调控的关系,为该病害的防治打下基础。【方法】以小麦品种扬麦12为试验材料,以异沙叶蝉为传毒介体饲毒后转移到1叶期的健康幼苗(3头/株)上进行传毒,同时以无毒异沙叶蝉取食健康幼苗为对照。根据试验需要,不同时间取样备用。为保证试验的准确性,经PCR检测为阳性的作为处理组试验材料;采用间接酶联免疫吸附(ELISA)法,利用植物赤霉素(GA3)试剂盒测定分析侵染第21天取样的小麦叶片赤霉素含量;将带毒条沙叶蝉接种的小麦苗分为两个平行处理组,接种后第7天分别用GA3(浓度为50 mg·L-1)和H2O进行叶面喷施处理,每隔一周处理一次。以无毒叶蝉接种后长势一致的小麦苗作为对照组,根据株高统计结果分析外施赤霉素对受侵染小麦植株的表型变化;以山羊草(Aegilops tauschii)的内根-贝壳杉烯合成酶(ent-kaurene synthase-like 3,KSL3)的基因编码区序列为参考基因设计引物(KSL3-F:5′-ATGATGGTGAATCCGCCGC-3′;KSL3-R:5′-TTAATGGTTGATCTTTGTTT-3′),对扬麦12的KSL3进行克隆和序列分析;分别取接种后7、14和21 d的小麦植株叶片,提取RNA后反转录,以克隆得到的Ta KSL3基因序列设计引物(Ta KSL3-F:5′-GAGACATGTGCCATGGCGTTC-3′;Ta KSL3-R:5′-CGTGTCACTCAGATCGGTGGAG-3′),选择小麦翻译延伸因子1A(EF-1α)作为内参基因,利用荧光定量PCR方法分析赤霉素代谢相关基因的转录水平。【结果】经ELISA检测发现,接种21 d的发病植株赤霉素含量与健康植株相比降低了28.9%;通过施用浓度为50 mg·L-1的赤霉素后,发病植株的平均株高相比对照组显著增加35.9%;采用同源克隆得到了完整的小麦赤霉素合成途径关键酶KSL3的编码区序列,长度为1 827 bp,编码608个氨基酸,BLAST比对分析发现该DNA序列与山羊草KSL3编码区序列相似度为85.2%。经荧光定量检测发现受小麦矮缩病毒侵染后小麦KSL3表达量显著下降,接种14 d降低为对照组的35.7%,21 d降低为对照组的9.6%。【结论】小麦矮缩病毒的侵染导致赤霉素合成途径关键酶的表达量降低,可能使赤霉素合成受阻,赤霉素含量降低引发受赤霉素调节的细胞生物学过程异常,从而诱导矮化症状形成。研究结果为揭示小麦矮缩病毒侵染的致病机理和病害防控打下了基础。     

棉花蔗糖合成酶基因家族在纤维发育期时空表达模式分析

蔗糖合成酶(Sucrose Synthase)是调控植物糖类代谢的关键酶之一,在植物生长发育及渗透调节过程中起着重要的作用。该酶为多基因家族,各成员在植物不同组织及不同发育阶段发挥不同的作用。陆地棉(Gossypium hirsutum)中有15个Sus成员,本研究利用转录组高通量测序及实时定量PCR技术,检测了该基因家族的表达模式。结果表明,Gh Sus1D、Gh Sus3A、Gh Sus3D和Gh Sus6D在起始期(0~3 DPA)大量表达;GhSus7D在10 DPA的纤维中表达量快速增加,达到同期突变体胚珠的4.7倍;Gh Sus1A、Gh Sus5D在纤维伸长期(5~15 DPA)表达量显著高于胚珠,分别在10 DPA、15 DPA时达到峰值;Gh Sus3A、Gh Sus3D、和Gh Sus6D在纤维发育(15 DPA)时表达量重新上升,且在20 DPA时仍处于较高表达水平。Sus基因家族组织表达模式分析表明,它们在陆地棉品种徐州142野生型和突变体中的组织表达模式基本相同,根、茎、叶、花中没有显著差异。但不同Sus基因具有不同的组织表达特异性。     

Changes in the properties of rice varieties with different amylose content on dry heat parboiling

Dry heat parboiling is a unique paddy processing technique that has been scarcely exploited. Dry heat parboiling at high temperature for short time and low temperature for long time on physical and physicochemical properties of three rice varieties differing in amylose content were studied. Hardness of the kernels increased from 66.4 N, 68.8 N and 59.8 N in raw samples to 89.1 N, 86.9 N and 59.8 N in parboiled high amylose, low amylose and waxy rice samples respectively. Rapid migration and evaporation of water from severely heated kernels caused cavity formation at the centre. Irreversible damage of amylopectin structures to leachable fractions caused continuous rise of the pasting curve. Crystallinity was thereby reduced. Parboiled high amylose samples gave X-ray diffraction patterns with peaks characteristic of A, B and V-type starch crystallinity. Crystalline starch-lipid complexes were observed in low amylose and waxy rices. The significant increase in the amount of rapidly digestible starch from 56.7%, 61.7% and 66.6% in raw samples to 92.1%, 90.8% and 94.8% respectively in severely processed rice samples and subsequent reduction in resistant starch from 24.5%, 21.2% and 18.4% to 0.4%, 1.9% and 0.1% indicated possibilities for targeted food use of the dry heat parboiled samples.     

柑橘CHS基因序列多态性及表达水平对类黄酮生物合成的影响

查尔酮的合成是类黄酮生物合成途径中的一个关键节点。从10个柑橘种质中克隆了查尔酮合成酶(chalcone synthase,CHS)基因,并对其不同时期的果实和叶片中的类黄酮含量进行了测定,分析了CHS基因的序列多态性与类黄酮含量之间的关系;同时通过qRT-PCR检测了CHS基因在不同种质之间以及同一种质的不同组织间的表达差异。结果表明,柑橘CHS基因的核苷酸序列高度保守,相似度达98%以上。聚类分析表明,CHS氨基酸序列的多态性有物种特异性,而且与类黄酮含量有一定的相关性。CHS基因的表达水平在不同种质、部位及生长发育时期有显著差异,这些差异与类黄酮的含量有显著的相关性,说明CHS基因对类黄酮的生物合成有明显影响。     

Wx^mp基因背景下可溶性淀粉合成酶基因SSⅡa和去分支酶基因PUL对水稻蒸煮食味品质的影响

【目的】分析在同一主效基因(Wx^mp)背景下可溶性淀粉合成酶基因SSⅡa和去分支酶基因PUL对稻米蒸煮食味品质的影响,以期为水稻品质遗传改良提供依据。【方法】选择在SSⅡa和PUL存在多态性而其他淀粉合成酶相关基因没有多态性的半糯品系宁0145和粳稻品种武运粳21进行杂交,获得F2群体与F3株系。利用分子标记,选择含有Wx^mp基因的F2单株与F3株系,将这些F2单株与F3株系分成SSⅡa^nPUL^n、SSⅡa^nPUL^w、SSⅡa^wPUL^n和SSⅡa^wPUL^w4种基因型(n和w分别表示该基因来源于宁0145和武运粳21),分析不同基因型蒸煮食味品质性状的差异,探讨同一Wxmp基因背景下不同SSⅡa和PUL等位基因对蒸煮食味品质性状的影响。【结果】不同基因型间蒸煮食味品质性状均存在显著差异,来源于武运粳21的SSⅡa^w基因和PUL^w基因分别使直链淀粉含量增加0.29%~1.00%和0.62%~1.18%,且PUL的效应大于SSⅡa,两者间存在互作效应。SSⅡa^w基因和PUL^w基因降低胶稠度和崩解值,提高了热浆黏度、冷胶黏度、消减值和回复值,对糊化温度、峰值黏度和峰值时间的作用较小。【结论】明确了Wx^mp背景下SSⅡa和PUL基因对稻米蒸煮食味品质的遗传效应,该研究结果为SSⅡa和PUL基因的分子标记辅助选择改良稻米品质提供了理论依据。     

Fusarium solani Upregulated Sesquiterpene Synthase Expression,Sesquiterpene Production and Allelopathic Activity in Piper betle L.

Activators of sesquiterpene synthase (STS) gene expression and sesquiterpene production in Piper betle L. were examined using quantitative real time PCR and gas chromatography mass spectrometry methods, and the allelopathic activity of untreated and Fusarium solani-treated betel extracts was tested on seed germination and on the shoot and root growth of Thai rice variety PSL2 (Oryza sativa cv. Phitsanulok 2) and three dominant paddy weeds (Eclipta prostrata, Echinochloa crus-galli and Chloris barbata). The results demonstrated that F. solani dramatically upregulated STS expression and productions of β-cubebene, β-caryophyllene and germacrene D sesquiterpene when compared with the untreated control, and that betel extracts had a greater inhibitory effect on weeds than on rice. The effects were more clearly detected on seed germination and root growth than on shoot growth, and they were found to be dose-dependent. It is also noted that F. solani-treated extract had stronger effects than the untreated extract. The species most sensitive to the allelopathic effects was C. barbata, germination of which was completely inhibited even at a dose of 0.1 mg/mL untreated extract. With regards to rice, although betel extract at 1.0 mg/mL showed no inhibition on germination, it affected the elongation of rice roots, in addition to those of the tested weeds. The obtained data suggested that F. solani has potential as an activator of sesquiterpene allelochemical production via STS expression, the latter leading to the treated betel extract having a stronger phytotoxic effect. These results were beneficial in the promotion of natural herbicide production using biotechnology.     

茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建

CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。