Cloning and expressional characterization of soybean GmLls1 gene

In higher plants, green leaf is a very important part involved in the photosynthesis process. Good develop-ment of the leaves is the guarantee of high yields and quality. Leaf senescence that occurs in the last stage of leaf development is a genetically p…     

RNAi-mediated knockingdown of rlpk2 gene retarded soybean leaf senescence

Leaf senescence that occurs in the last stage of leaf development is a genetically programmed process. It is very significant to elucidate the molecular mechanisms that control the initiation and progression of leaf senescence and the way the senescence signal is transduced. In a previous study on artificially induced soybean leaf senescence, we cloned a novel gene designated rlpk2 (Genbank Accession No. AY687391) that encodes a leucine-rich repeat (LRR) receptor like protein kinase. The expression level of rlpk2 gene was shown to be strongly up-regulated during both the natural leaf senescence process in this report and the artificially induced primary-leaf-senescence process in our previous work. The RNA interference (RNAi)-mediated knocking-down of rlpk2 dramatically retarded both the natural and nutrient deficiency-induced leaf senescence in transgenic soybean. The transgenic leaves showed more cell-aggregated surface structure and higher content of chlorophyll.     

大豆叶片衰老过程中半胱氨酸蛋白酶基因的特异性表达

用反转录和多聚酶链反应（ＲＴ－ＰＣＲ）和琼脂糖凝胶电泳方法对大豆叶片衰老过程中半胱氨酸蛋白酶基因的表达情况进行了研究，发现由ＰＣＲ扩增出的ｃＤＮＡ带谱会随着叶片的不断衰老而发生较明显的改变，初步证明，至少有一条ｃＤＮＡ带为绿叶样品所特有，另外，两条ｃＤＮＡ带则为衰老叶片样品所特有，该实验结果为进一步分离大豆叶片衰老时特异表达基因及其启动子和最终实施转基因工程奠定了基础。     

Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. Tabaci

LSD1-related proteins of Arabidopsis with LSDl-like zinc finger domains regulate disease resistance and programmed cell death （PCD）. We cloned a rice OsLOL2 gene, orthologous to LSD1 of Arabidopsis and expressed it in a tobacco plant. Transgenic tobacco lines displayed enhanced disease resistance to a virulent bacterium Pseudomonas syringae pv. tabaci （Pst）. RT-PCR analysis showed that overexpression of OsLOL2 in transgenic tobacco lines resulted in upregulation of two pathogenesis-related （PR） protein genes, PR2 and PR5. Our results suggest that overexpression of OsLOL2 in transgenic tobacco enhances the resistance through the induction of PR proteins and hypersensitive response-like reaction.     

An <Emphasis Type="Italic">Arabidopsis ctpA</Emphasis> homologue is involved in the repair of photosystem II under high light

A T-DNA insertion mutant AtctpA1 was identified to study the physiological roles of a carboxyl-terminal processing protease （CtpA） homologue in Arabidopsis. Under normal growth conditions, disruption of AtctpA1 did not result in any apparent alterations in growth rate and thylakoid membrane protein components. However the mutant plants exhibited increased sensitivity to high irradiance. Degradation of PSII reaction center protein D1 was accelerated in the mutant during photoinhibition. These results demostrated that AtctpA1 was required for efficient repair of PSII in Arabidopsis under high irradiance.     

Isolation and characterization of soybean NBS analogs

Isolation of plant resistance genes is greatly helpful to crop resistance breeding and the insight of resistance mechanism. The cloned plant resistance genes are classified into four classes according to their putative structural domain, of which the majority possesses nucleotide-binding site (NBS) domain that consists of P-loop, kinase2a and kinase3a. The conservation of this domain affords the potential possibility of cloning the plant resistance genes, which is homology-based cloning technique. In the present study, the degenerate oligonucleotide primers were designed according to the tobaccoN andArabidopsis RPS2, and 358 clones were isolated from the genomic DNA of resistance soybean cultivar Kefengl, resistant to soybean mosaic virus, and 4 open-reading NBS analogs were finally characterized and designated asKNBS1, KNBS2, KNBS3 andKNBS4. Southern hybridization suggested that they were present with multicopy in the soybean genome;KNBS4 was mapped to F linkage group andKNBS2 co-located J linkage group with the SCAR marker ofRsa resistant to soybean mosaic virus by RFLP analysis. Northern analysis suggested thatKNBS2- related sequence was low and constitutively expressed in the root, stem and leaves of soybean. The detailed characterization of NBS analogs is very helpful to ultimately cloning the soybean resistance gene.     

A wound-induced small polypeptide gene family is upregulated in soybean nodules

Small peptides function as key signals in processes, such as plant cell differentiation, organ development and defenses to biotic stresses. A large number of small peptide precursor genes have been predicted from the analysis of the soybean (Glycine max) whole genome DNA sequence. However, most of these genes have unknown characteristics and functions. In this report, we systemically searched for the gene families of small peptide precursors that are up-regulated in soybean nitrogen-fixing root nodules. We found 212 genes (encoding peptides shorter than 150 amino acids) that were up-regulated, and among them, 79 genes belong to 38 multiple-gene families, but the other 133 genes are unique. Twenty-eight of 38 families are conserved in Arabidopsis, but the other 10 only exist in legumes. We also identified 16 out of the 38 members of the wound-induced polypeptide (WIP) gene family to be upregulated in nitrogen-fixing nodules. We further analyzed homologs of WIP genes in Medicago, Lotus, Arabidopsis and Oryza species and found that a few homologous genes from Medicago truncatula and Lotus japonicus were also upregulated in their nodules and some WIP genes were induced by specific fungal pathogens on soybean and rice. Structure prediction indicated that all WIP prepropeptides contain a conserved DUF3774 domain (including two hydrophobic regions) and most of them have an N-terminal signal sequence. Fluorescence microscopy analysis of two WIP prepropeptides fused to GFP revealed that these proteins are located on the plasma membrane of tobacco leaf cells. Interestingly, 34 soybean WIP genes are clustered onto three soybean chromosomes, different from known peptide gene families (such as CLE). Among them, 11 highly identical genes are aligned on the 6th chromosome, 12 on the 12th, and 11 on the 13th chromosomes. Most of WIP genes from the 12th chromosome share the highest identities with their homologs on the 13th chromosome, suggesting that ancestral WIP genes could have originated from the 13th chromosome, then spread onto the 12th chromosome by chromosome homologous recombination; the new WIP genes could have existed in multiple copies by gene duplication which then spread onto the 6th chromosome. In Arabidopsis and Oryza species, half of the WIP genes are also aligned on one chromosome and showed higher identity with those from the soybean 12th and 13th chromosomes, suggesting that WIP genes originated from one common ancestor.     

Transformation of japonica rice with<Emphasis Type="Italic">RHL</Emphasis> gene and salt tolerance of the transgenic rice plant

Overexpression of the yeastHAL2 gene increases salt tolerance of yeast and plant. RiceHAL2-like (RHL) gene was introduced into ajaponica rice cultivar HJ19 withAgrobacterium tumefaciens-mediated transformation. Transgenic plants in R₀ generation were selected on the principle of GUS-positive,RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R₁ generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R₁ generation showed that theRHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspensioncultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was discussed.     

Molecular characterization of RAPD and SCAR marker linked to the frog-eye leaf spot resistance gene in soybean

Two fragments SCS3₆₂₀ and SCS3₅₈₀ of the co-dominant marker OPS03_{620 & 580} that were linked to the resistance gene of soybean frog-eye leaf spot have been completely sequenced. A significant insertion of 30 bp is the main reason of the polymorphism between the two fragments. The results of Southern hybridization indicate that SCS3₆₂₀ derives from a single-or low-copy sequence and can be used as an RFLP probe. A co-dominant SCAR marker SCS3_{620 & 580} has been developed based on the sequences. The segregation of SCS3_{620 & 580} is similar to that of RAPD marker OPS03_{620 & 580}-Significant polymorphism has been shown between resistant and susceptible genotypes when 62 soybean genotypes were surveyed for the SCAR marker. Therefore, the marker can be used in the resistance breeding of soybean frog-eye leaf spot by marker-assisted selection.     

栽培密度对有色稻抽穗后剑叶一些衰老生理特性的影响

以黑糯和红粳两个有色稻为材料,设5种不同的栽培密度,研究了不同栽培密度对有色稻抽穗后剑叶的叶绿素含量、可溶性蛋白质含量和SOD酶活性等一些衰老生理指标的影响。结果表明：随栽培密度的增加,有色稻抽穗后剑叶叶绿素a、b含量、可溶性蛋白质含量和SOD酶活性均呈下降趋势,而叶绿素a/b值和丙二醛含量呈上升趋势,且高密度E处理下这些指标变化幅度较大,衰老加快。试验还表明,有色稻抽穗后剑叶叶绿素含量、SOD酶活性、丙二醛含量等衰老生理指标的变化幅度与品种的耐密性密切相关。     

金花茶回归苗木的生长、光合特性和叶片形态特征

金花茶（Camellia nitidissima）为国家二级重点保护野生植物,具有重要的观赏、药用和科研价值,但其野生资源遭到极为严重的破坏,有濒临灭绝的风险,因此开展金花茶的回归引种,可有效保护该物种。为探究金花茶实生苗、扦插苗回归引种到原生境近7年后在生长和光合生理特性方面的差异,本研究对其生长状况、成活率、光合特性和叶片形态特征等进行测定。结果表明：金花茶回归苗木实生苗比扦插苗具有更大的生长量和更高的成活率;实生苗最大净光合速率（P_max）、光饱和点（LSP）、表观量子效率（AQY）均显著高于对应点的扦插苗（P<0.05）,其光能利用范围也大于对应点的扦插苗;实生苗的叶绿素a （Chl a）、叶绿素b （Chl b）和类胡萝卜素（Car）含量均显著低于对应点的扦插苗（P<0.05）,但叶片厚度、海绵组织厚度、中脉导管直径和叶面积则均显著大于对应点的扦插苗（P<0.05）;双因素方差分析结果表明,回归点和苗木类型分别对金花茶的株高、地径、成活率、P_max、LSP、叶绿素含量和叶面积均有极显著影响（P<0.01）。因此,在开展金花茶回归引种中宜优先选用实生苗种植,并选择具有中等遮阴环境的野外生境进行回归。     

2，4-二叔丁基苯酚对细叶小羽藓生理的影响

为探究木麻黄（Casuarina equisetifolia）林地化感物质中含量最为丰富的2，4-二叔丁基苯酚（2，4-di-tert-butylphenol，2，4-DTBP）对细叶小羽藓（Haplocladium microphyllum）生长发育的影响，用不同物质的量浓度（0，0.01，0.05，0.10，0.50，1.00 mmol·L^-1）的2，4-DTBP处理细叶小羽藓，分别进行叶绿素、脯氨酸和丙二醛（MDA）含量的测定.结果表明：用适宜物质的量浓度（0~0.10 mmol·L^-1）2，4-DTBP处理时，细叶小羽藓叶绿素含量增加，脯氨酸和丙二醛含量下降；而物质的量浓度大于0.50 mmol·L^-1时，叶绿素含量下降，脯氨酸和丙二醛含量增加.当2，4-DTBP浓度高于0.50 mmol·L^-1时，对细叶小羽藓产生明显的化感抑制效应，不利于细叶小羽藓生长.本研究以常见种细叶小羽藓为例，探讨了2，4-DTBP对苔藓植物的化感作用，为细叶小羽藓是否适应木麻黄林地生态环境提供参考.     

Functional analysis of cis-acting sequences regulating root-specific expression in transgenic tobacco

Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-specific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.     

The foxtail millet Si69 gene is a Wali7 (wheat aluminum-induced protein 7) homologue and may function in aluminum tolerance

We isolated a clone, named Si69, from a foxtail millet immature seed cDNA library. The protein encoded by Si69 contains a conserved Wali7 (wheat aluminum induced protein 7) domain and shares high-level homology with aluminum-induced proteins from other species including rice and Arabidopsis. The Si69 gene presents as a single locus in foxtail millet genome and is globally expressed in all tissues examined. Its expression is up-regulated by aluminum. The sequence feature and expression pattern suggest that the Si69 gene is involved in aluminum tolerance or detoxification. To confirm its biological functions, Si69 controlled by the CaMV35S promoter was introduced into Arabidopsis. Transgenic plants did not show any visible morphological changes compared to wild-type plants under normal growth conditions. However, when treated with 20 or 50 μmol/L Aluminum (Al), the root apices of wild-type plants were heavily stained by hematoxylin, whereas those of Si69 transgenic plants were not stained when treated with 20 μmol/L Al and slightly stained when treated with 50 μmol/L Al. Scanning electron microscopy (SEM) results further demonstrated that the damage of the root apices was severer in wild-type plants than in transgenic plants. Inhibition of root growth and accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation, were lower in transgenic plants than in wild-type plants. The results show that overexpression of Si69 may increase Al tolerance in transgenic plants, indicating that a series of Wali7-containing genes may play similar roles in Al tolerance/detoxification.     

盐胁迫对红鳞蒲桃苗木生长和生理特性的影响

【目的】评价盐胁迫下红鳞蒲桃(Syzygizan hancei)苗木的生理耐受性。【方法】选择当年生红鳞蒲桃盆栽苗木为材料,研究6种NaCl盐分浓度(0%、0.2%、0.4%、0.6%、0.8%、1.0%)下红鳞蒲桃幼苗高生长、地径生长、叶片质膜透性、游离脯氨酸含量、可溶性糖含量、过氧化氢酶(CAT)活性、叶绿素含量的变化特征。【结果】随着NaCl浓度的增加,红鳞蒲桃苗木的高增长量逐渐降低,在浓度0.4%时下降显著;NaCl浓度对苗木的地径增长量影响不大;细胞质膜透性对盐胁迫具有较强的耐性,NaCl浓度在0.8%以下时,叶片的相对电导率差异不显著;游离脯氨酸和可溶性糖含量在NaCl浓度为0.4%时显著增加,其后维持在一定水平;CAT酶活性对盐胁迫较为敏感,在浓度0.2%时即显著增强;叶绿素含量则随着NaCl浓度的增加表现出先升后降的变化趋势。【结论】0.4%NaCl浓度是影响红鳞蒲桃苗木生理特性的关键。     

萘对铜绿微囊藻和聚球藻生长及叶绿素荧光影响的比较

为探究萘对不同粒径蓝藻的胁迫效应,采用5组不同质量浓度萘(0、0.01 mg/L、0.1 mg/L、1 mg/L、10 mg/L)分别对聚球藻(Synechococcus sp.)和铜绿微囊藻(Microcystis aeruginosa,PCC7806)进行为期7 d的处理,分析不同浓度萘处理下,藻细胞浓度、叶绿素a含量及叶绿素荧光参数的变化,探究不同浓度萘处理对2种藻的生长及生理影响。结果表明:(a)整个处理周期内,聚球藻的生长被显著抑制,其叶绿素a含量减小了3.9%~40.4%;(b)聚球藻的潜在光合作用能力、光合作用速率均有所减弱,但其耐受强光的能力有所增强,说明聚球藻对萘具有较强的敏感性;(c)萘对铜绿微囊藻生长具有显著的促进作用,增强了其潜在光合作用能力,但抑制了其细胞内叶绿素a的合成。     

Reporter-based screen for <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants compromised in nonhost resistance

Plants are exposed to many potentially pathogenic microbes in the environment, but each species is only susceptible to a limited number of pathogens. The broad resistance is referred to as nonhost resistance. To date, little is known about the underlying mechanism of nonhost resistance and the signaling transduction process. Here we describe a simple method for isolating Arabidopsis nonhost resistance mutants against a nonadapted bacterial pathogen. A RAP2.6 promoter-driven LUC reporter system was developed to replace the tedious bacterial growth assay during the primary screening. The RAP2.6-LUC reporter gene is normally induced by the virulent bacterium Pseudomonas syringae pv tomato but not the nonadapted bacterium P. syringae pv phaseolicola. By using this method we iso- lated 4 mutants displaying strong reporter activity in response to P. syringae pv phaseolicola, which were characterized in some details, ebsl, ebs2, ebs3, and ebs4 （enhanced bacterial susceptibility） were compromised in resistance against P. syringae pv phaseolicola and/or P. syringae pv tomato. In addition, ebs4 showed enhanced hypersensitive response to the incompatible bacterium P. syringae pv tomato （avrB）. These results demonstrated that the method is suited for large scale screening for nonhost resistance mutants.