Cloning of TaCYP707A1 Gene that Encodes ABA 8′-Hydroxylase in Common Wheat (Triticum aestivum L.)

The plant hormone abscisic acid （ABA） regulates many important physiological and developmental processes in plants. The objective of this study was to clone the ABA 8′-hydroxylase gene in common wheat. In the present study, we used the eDNA sequence of barley HvCYP707A1 gene （GenBank accession no. AB239299） as a probe for BLAST search against the common wheat （Triticum aestivum L.） EST database in GenBank. All wheat ESTs sharing high similarity with the reference gene were subjected to contig assembly. Primers were designed based on the constructed contigs to clone the wheat CYP707A1 gene, designated as TaCYP707A1. The genomic DNA sequence of TaCYPTO7A1 gene comprised five exons and four introns, with a size of 2225 bp. The corresponding cDNA sequence of TaCYP707A1 was 1737 bp, containing an open reading frame （ORF） of 1431 bp, a 42-bp 5′-untranslated region （UTR） and a 264-bp 3′UTR, with 94.9% of identical sequences to HvCYP707A1 gene （AB239299）. The neighbor joining tree indicated that the deduced amino acid sequences of TaCYP707A1 gene was highly similar to those of barley and rice. The TaCYP707A1 gene was located on chromosome 6BL using a set of Chinese Spring nullisomic-tetrasomic lines and ditelosomic line 6BS. These results will be of high importance in understanding of molecular mechanism of ABA catabolism.

Transcriptional Analysis of 9-Cis-Epoxycarotenoid Dioxygenase, Glucosyltransferase, 8＇-Hydroxylase and B-Glucosidase Genes that Regulate Abscisic Acid Homeostasis around the Onset of Grape Berry Ripening

The mechanisms for fine-tuning ABA level related to grape berry ripening remain elusive. Here, transcription analysis showed that the mRNA expression level of 9-cis-epoxycarotenoid dioxygenase gene （VvNCED1） increases first, rapidly in mesocarp before the onset of grape berry ripening. After VvNCED1 peaks its expression level, ABA content increases rapidly in mesocarp coupled with an increase in both soluble sugar content and pH value. On the onset of berry ripening, VvNCED1 transcripts decline rapidly to its lowest point, then increases slightly. Whereas, the mRNA expression level of B-glucosidase gene VvBGI, on the whole, increases constantly during grape berry ripening. During berry de-greening, ABA glucosyltransferase （VvGT） and ABA 8＇-hydroxylase （VvCYPI） equilibrate ABA level; during berry coloring-up, VvGT predominantly equilibrates ABA level, namely, the up-regulation of ABA level mainly leads from VvNCED1 and VvBG1 gene high expression; the down-regulation of ABA level leads mainly from VvCYP! transcript level both in ABA content- and developmental phase-dependence manner. In conclusion, our main results show that VvNCED1 and VvBG1 genes are closely related to grape berry ripening.

小麦TaPYL8基因的克隆与抗旱功能分析

脱落酸(ABA)信号通路在植物对干旱胁迫的响应中扮演着至关重要的角色;作为ABA受体,PYL家族蛋白在ABA信号转导中发挥核心作用。小麦TaPYL8是PYL家族的成员。为了明确TaPYL8是否参与小麦干旱胁迫响应,本研究分析了TaPYL8的表达模式,创建过表达TaPYL8拟南芥,并探讨了干旱对拟南芥生理生化及胁迫相关基因表达的影响。结果表明,TaPYL8表达量在干旱胁迫、外源ABA及PEG6000处理后上调。干旱胁迫下,过表达TaPYL8增强了拟南芥的存活率,降低了叶片失水率和MDA含量,提高了SOD、POD和CAT等抗氧化酶活性及AtSOD、AtP5CS1、AtABF3等胁迫相关基因的表达量。推测TaPYL8通过促进胁迫相关基因表达增强植物抗旱能力。

ABA分解代谢及其代谢关键酶—8′-羟化酶

脱落酸(ABA)在植物的生长发育和环境胁迫响应等过程中具有重要作用。ABA合成与分解代谢的动态平衡共同调控植物内源ABA水平。ABA8′位甲基羟基化途径是高等植物内源ABA代谢的主要途径;8′-羟化酶是该代谢途径的关键酶,属于P450酶系。生物化学和基因组学研究表明,拟南芥CYP707A家族基因编码8′-羟化酶,该基因家族广泛存在于高等植物中,调控植物内源ABA代谢,介导ABA相关的生理生化过程。本文综述了ABA分解代谢的基本途径,详细概述了ABA8′位甲基羟基化途径及该代谢途径的关键酶8′-羟化酶。同时介绍了8′-羟化酶编码基因-CYP707A家族基因的生物学特征和功能。

RNAi-mediated suppression of the abscisic acid catabolism gene Os ABA8ox1 increases abscisic acid content and tolerance to saline–alkaline stress in rice(Oryza sativa L.)

Saline–alkaline(SA) stress is characterized by high salinity and high alkalinity(high p H), which severely inhibit plant growth and cause huge losses in crop yields worldwide. Here we show that a moderate elevation of endogenous abscisic acid(ABA) levels by RNAi-mediated suppression of Os ABA8 ox1(Os ABA8 ox1-kd), a key ABA catabolic gene, significantly increased tolerance to SA stress in rice plants. We produced Os ABA8 ox1-kd lines in two different japonica cultivars, Dongdao 4 and Nipponbare. Compared with nontransgenic control plants(WT), the Os ABA8 ox1-kd seedlings accumulated 25.9%–55.7% higher levels of endogenous ABA and exhibited reduced plasmalemma injury, ROS accumulation and Na;/K;ratio, and higher survival rates, under hydroponic alkaline conditions simulated by 10, 15, and 20 mmol L-1 of Na;CO;. In pot trials using SA field soils of different alkali levels(p H 7.59, 8.86, and 9.29), Os ABA8 ox1–kd plants showed markedly higher seedling survival rates and more vigorous plant growth, resulting in significantly higher yield components including panicle number(85.7%–128.6%), spikelets per panicle(36.9%–61.9%), branches(153.9%–236.7%), 1000–kernel weight(20.0%–28.6%), and percentage of filled spikelets(96.6%–1340.8%) at harvest time. Under severe SA soil conditions(p H = 9.29, EC = 834.4 μS cm-1),Os ABA8 ox1-kd lines showed an 194.5%–1090.8% increase in grain yield per plant relative to WT plants.These results suggest that suppression of Os ABA8 ox1 to increase endogenous ABA levels provides a new molecular approach for improving rice yield in SA paddies.

OsABA8ox2, an ABA catabolic gene, suppresses root elongation of rice seedlings and contributes to drought response

In rice, OsABA8ox encodes abscisic acid(ABA) 8′-hydroxylase, which catalyzes the committed step of ABA catabolism. The contribution of ABA catabolism in root development remains unclear. We investigated the role of OsABA8ox2 in root growth and development and drought response. GUS staining results showed that OsABA8ox2 was expressed mainly in roots at seedling stage and was strongly expressed in the meristematic zone of the radicle. OsABA8ox2 expression in roots was markedly decreased after 0.5 h polyethylene glycol(PEG) treatment and increased after 0.5 h rehydration, implying that OsABA8ox2 is a drought-responsive gene.OsABA8ox2 knockout mediated by the CRISPR-Cas9 system increased drought-induced ABA and indole-3-acetic acid accumulation in roots, conferred increased ABA sensitivity, and promoted a more vertically oriented root system architecture(RSA) beneficial to drought tolerance.OsABA8ox2 overexpression suppressed root elongation and increased stomatal conductance and transpiration rate. Consequently, OsABA8ox2 knockout dramatically improved rice drought tolerance, whereas OsABA8ox2 overexpression seedlings were hypersensitive to drought stress,suggesting that OsABA8ox2 contributes to drought response in rice. Compared with wild type,functional leaves of OsABA8ox2 knockout seedlings showed higher ABA levels, whereas overexpression lines showed lower ABA levels, suggesting that OsABA8ox2, as an ABA catabolic gene, modulates ABA concentration through ABA catabolism. OsABA8ox2 and OsABA8ox3 were both localized in the endoplasmic reticulum. Together, these results indicate that OsABA8ox2 suppresses root elongation of rice seedlings, increases water transpiration, and contributes to drought response through ABA catabolism, and that OsABA8ox2 knockout dramatically improves rice drought tolerance. They highlight the key role of ABA catabolism mediated by OsABA8ox2 on root growth and development. OsABA8ox2, as a novel RSA gene, would be a potential genetic target for the improvement of rice drought tolerance.

An efficient corrosion inhibitor of cassava starch graft copolymer for aluminum in phosphoric acid

Starch is one of the richest natural polymers with low-cost,non-toxic and biodegradable,but is seldom directly used as corrosion inhibitor due to its poor inhibitive ability and low water solubility.To solve this problem,cassava starch-acryl amide graft copolymer(CS-AAGC)was prepared through grafting acryl amide(AA)with cassava starch(CS),and it was firstly examined as an efficient inhibitor for 1060 aluminum in 1.0 mol·L^(-1)H_(3)PO_(4) media.The adsorption behavior of CS-AAGC and its electrochemical mechanism were investigated by weight loss and electrochemical methods.Additionally,the inhibited aluminum surface was fully characterized by a series of SEM,AFM,contact angle measurements and XPS.Results confirm that CS-AAGC performs better inhibitive ability than CS,AA or CS/AA mixture,and the maximum inhibition efficiency of 1.0 g·L^(-1)CS-AAGC is 90.6%at 20℃.CS-AAGC acts as a mixed-type inhibitor while mainly retards the anodic reaction.EIS has three time constants,and the polarization resistance is significantly increased in the presence of CS-AAGC.The micrograph of inhibited aluminum surface is of hydrophobic nature with low surface roughness and little corrosion degree.

High efficient removal of HCN over porous CuO/CeO2 micro-nano spheres at lower temperature range

The porous CeO2 flowerlike micro-nano spheres based materials were prepared to remove HCN effectively at lower temperature range.The CeO2 and a serious of porous flowerlike ceria based materials loaded with metal species including Cu,Ag,Ni,Co and Fe were synthesized by hydrothermal method and precipitation method respectively.The physicochemical properties were probed by means of XRD,H2-TPR,BET,SEM and XPS.The removal ability for 130 mg·m﹣3 HCN over CuO/CeO2 showed the highest activity,the breakthrough time of which was more than 70 min at the condition of 30℃,120,000 h-1 and b 5%(volume)H2O,owe to a higher relative atomic ratio of oxygen vacancies and Oβ,the stronger interaction between metal particle and support,the optimum redox properties.The reaction mechanism was speculated by detecting the reaction products selectivity at different reaction temperature.It was shown that the reaction system for removal of HCN over CuO/CeO2 catalytic material involved chemisorption,catalytic hydrolysis,catalytic oxidation as well as NH3-SCR reactions.

OsbZIP09,a Unique OsbZIP Transcription Factor of Rice,Promotes Rather Than Suppresses Seed Germination by Attenuating Abscisic Acid Pathway

We successfully identified a novel and unique OsbZIP transcription factor,OsbZIP09,whose mutants exhibited longer seeds and less severe pre-harvest sprouting than the wild type,but shared similar germination rate as the wild type under normal germination conditions.The expression of OsbZIP09 was induced by abscisic acid(ABA)and declined as the germination process.As a nucleus-localized transcription factor,the conserved binding motif of OsbZIP09 was identified via DNA affinity purification sequencing technique.Further evidences indicated that OsbZIP09 directly enhanced the expression of ABA catabolism gene ABA8ox1,thus reducing ABA accumulation.In addition,OsbZIP09 also directly bound to the promoter of LEA3 gene to inhibit its expression,thus further alleviating the suppressive effect of ABA on seed germination.These results demonstrated that OsbZIP09 likely functions as a brake of the ABA pathway to attenuate the inhibitory effect of ABA on rice seed germination via dual strategies.

太子参分解代谢关键酶8'羟化酶基因的克隆及生物信息学分析

ABA 8＇羟化酶是脱落酸（abscisic acid,ABA）分解代谢中的关键酶基因之一。采用同源检索的方法从太子参转录组数据库中获得7个ABA 8＇羟化酶（abscisic acid 8＇-hydroxylase）基因家族成员,对各家族成员进行转录分析组数据以及ABA 8＇羟化酶关键基因ABA8ox1编码区（coding sequence,CDS）的克隆与生物信息学分析。ABA8ox1基因CDS全长1 401 bp,编码480个氨基酸残基,等电点为8.55,相对分子质量为53 k Da,跨膜分析显示其为膜蛋白,1-21个氨基酸残基位于胞内,22-466个氨基酸残基位于胞外。转录组数据分析及定量PCR技术共同证明ABA8ox1在块根的韧皮部及须根中有较高的表达。多序列比对及聚类分析显示与其他植物CYP707A蛋白具有较高的同源性,且与模式植物拟南芥中ABA分解关键基因At CYP707A1和At CYP707A3聚为一类。该研究为太子参块根膨大及对逆境胁迫响应的分子机制奠定基础。

Two CYP82D Enzymes Function as Flavone Hydroxylases in the Biosynthesis of Root-Specific 4＇-Deoxyflavones in Scutellaria baicalensis

Baicalein, wogonin, and their glycosides are major bioactive compounds found in the medicinal plant Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no effect on normal cells. Furthermore, they have many additional benefits for human health, such as antioxidant, antiviral, and liver-protective properties. Here, we report the isolation and characterization of two CYP450 enzymes, SbCYP82D1.1 and SbCYP82D2, which function as the flavone 6-hydroxylase （F6H） and flavone 8-hydroxylase （F8H）, respectively, in S. baicalensis. SbCYP82D1.1 has broad substrate speci- ficity for flavones such as chrysin and apigenin and is responsible for biosynthesis of baicalein and scutel- larein in roots and aerial parts of S. baicalensis, respectively. When the expression of SbCYP82D1.1 is knocked down, baicalin and baicalein levels are reduced significantly while chrysin glycosides accumulate in hairy roots. SbCYP82D2 is an F8H with high substrate specificity, accepting only chrysin as its substrate to produce norwogonin, although minor 6-hydroxylation activity can also be detected. Phylogenetic analysis suggested that SbCYP82D2 might have evolved from SbCYP82D1.1 via gene duplication followed by neofunctionalization, whereby the ancestral F6H activity is partially retained in the derived SbCYP82D2.

桃CYP707A家族基因的克隆以及表达分析

从油桃‘中油四号’中克隆了3个CYP707A家族成员(PpCYP707A1、PpCYP707A2和PpCYP707A3)的全长,测序发现其CDS序列与桃基因组公布的序列一致。同源比对发现CYP707A家族在不同物种间具有高度的序列保守性,并且都含有CYP707A家族基因比较保守的血红素铁配位体半胱氨酸残基;系统进化树分析表明,PpCYP707A1与马铃薯CYP707A1、花生CYP707A1、菜豆CYP707A1、菜豆CYP707A2的亲缘关系较近,桃CYP707A2与拟南芥CYP707A2的亲缘关系较近,桃CYP707A3与拟南芥CYP707A4、花生CYP707A2的亲缘关系较近。荧光定量PCR分析表明,PpCYP707A1在茎中的表达量较高,PpCYP707A2在叶中的表达量较高,PpCYP707A3在花中的表达量较高,并且桃CYP707A家族一般在成熟组织中的表达水平要高于幼嫩组织。推测桃CYP707A家族成员交叉调控花芽休眠进程,并且发现花芽中的PpCYP707A2可以被ABA和GA诱导。原核诱导并纯化PpCYP707A1和PpCYP707A2蛋白可以为后续蛋白的功能鉴定奠定基础。