苏云金芽胞杆菌高效表达载体的构建

[目的]通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性,筛选出一个强启动子,利用强启动子构建一个苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)高效表达载体.[方法]利用启动子融合lacZ技术检测了4种启动子的转录活性.通过扫描电子显微镜观察晶体、SDS-PAGE、蛋白定量和生物活性测定等方法对新建高效表达载体进行功能验证.[结果]构建了Pcry1A、Pcry3A、Pcry4A和Pcry8E4个启动子融合报告基因lacZ的表达载体,经β-半乳糖苷酶活性分析得知,启动子活性从高到低依次为Pcry8E＞Pcry1A＞Pcry4A＞Pcry3A.选取cry8E启动子,以pHT315作为基础载体构建苏云金芽胞杆菌高效表达载体pHT315-8E21b,将cry1Ac基因连接到pHT315-8E21b和广泛应用的cry3A启动子指导的pSXY-422b上,分别转入无晶体突变株HD-73-,获得菌株HD-8E1Ac和HD-422-1Ac.扫描电子显微镜观察显示,HD-8E1Ac菌株可以形成菱形晶体,说明正确表达了cry1Ac基因.SDS-PAGE分析结合蛋白定量实验表明pHT315-8E21b表达效率高于pSXY-422b.对小菜蛾(Plutella xylostella)的生物活性测定表明HD-8E1Ac菌株对小菜蛾有生物活性,且菌株活性高于HD-422-1Ac.[结论]利用强启动子Pcry8E构建了一个能在Bt中高效表达的穿梭载体pHT315-8E21b,该载体可正确表达cry1Ac基因,其表达效率高于被广泛应用的pSXY422b.     

Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.

Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in β-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC₅₀ value was 0.0103 μg.mL^?1. The two mutations in β-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.     

Pyramiding Bt genes for increasing resistance of cotton to two major lepidopteran pests: Spodoptera litura and Heliothis armigera

To more effectively control two major cotton insects (cotton bollworm and Spodoptera litura) and improve the efficacy of the pest resistance management, novel transgenic plants expressing Bacillus thuringiensis Cry9C gene were generated, and gene stacking strategy was incorporated. Initially, a binary plasmid vector harboring Cry9C gene was introduced into an elite cotton cultivar Simian-3 by Agrobacterium-mediated transformation. Integration and expression of the Cry9C genes in three transgenic lines were confirmed by PCR and RT-PCR. Among these transgenic lines, T0 generation of line 16 (L-16) with normal phenotypes were selected by ELISA assays for its highest expression level of Cry9C. In T1 population of L-16, the expression level of Cry9C ranged from 29 to 45 μg/g fresh leaf. The following insect bioassays demonstrated that transgenic S3-35S::Cry9C cotton plants exhibited moderate toxicity to Heliothis armigera but strong toxicity to S. litura compared with the transgenic plants expressing Cry 1Ac gene. For incorporation of gene staking strategy, Cry9C gene and Cry 2A or Cry 1Ac were pyramided, respectively by sexual crossing. The expression of Cry9C protein in all F1 progenies had a similar level as the parent plants indicating the high heritability of Bt genes in transgenic progenies. Progenies from both Cry9C × Cry 2A and Cry9C × Cry 1Ac exhibited higher resistance to S. litura compared with their parents. Together our data demonstrated that our newly generated transgenic plants represent a reservoir of novel insect-resistant materials in cotton breeding, and the successful incorporation of gene pyramiding technology can provide a new solution of developing multiple resistance management strategies.     

Identification of medaka magnetoreceptor and cryptochromes

Magnetoreception is a hallmark ability of animals for orientation and migration via sensing and utilizing geomagnetic fields. Magnetoreceptor (MagR) and cryptochromes (Cry) have recently been identified as the basis for magnetoreception in Drosophila. However, it has remained unknown whether MagR and Cry have conserved roles in diverse animals. Here we report the identification and expression of magr and cry genes in the fish medaka (Oryzias latipes). Cloning and sequencing identified a single magr gene, four cry genes and one cry-like gene in medaka. By sequence alignment, chromosomal synteny and gene structure analysis, medaka cry2 and magr were found to be the orthologs of human Cry2 and Magr, with cry1aa and cry1ab being coorthologs of human Cry1. Therefore, magr and cry2 have remained as single copy genes, whereas cry1 has undergone two rounds of gene duplication in medaka. Interestingly, magr and cry genes were detected in various stages throughout embryogenesis and displayed ubiquitous expression in adult organs rather than specific or preferential expression in neural organs such as brain and eye. Importantly, magr knockdown by morpholino did not produce visible abnormality in developing embryos, pointing to the possibility of producing viable magr knockouts in medaka as a vertebrate model for magnet biology.     

<Emphasis Type="Italic">In vivo</Emphasis> fluorescence observation of parasporal inclusion formation in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A recombinant gene expressing a Cry1Ac-GFP fusion protein with a molecular mass of approximately 160 kD was constructed to investigate the expression of cry1Ac, the localization of its gene product Cry1Ac, and its role in crystal development in Bacillus thuringiensis. The cry1Ac-gfp fusion gene under the control of the cry1Ac promoter was cloned into the plasmid pHT304, and this construct was designated pHTcry1Ac-gfp. pHTcry1Ac-gfp was transformed into the crystal-negative strain, HD-73 cry⁻, and the resulting strain was named HD-73⁻(pHTcry1Ac-gfp). The gfp gene was then inserted into the large HD-73 endogenous plasmid pHT73 and fused with the 3′ terminal of the cry1Ac gene by homologous recombination, yielding HD-73Φ(cry1Ac-gfp)3534. Laser confocal microscopy and Western blot analyses showed for the first time that the Cry1Ac-GFP fusion proteins in both HD-73⁻(pHTcry1Ac-gfp) and HD-73Φ(cry1Ac-gfp)3534 were produced during asymmetric septum formation. Surprisingly, the Cry1Ac-GFP fusion protein showed polarity and was located near the septa in both strains. There was no significant difference between Cry1Ac-GFP and Cry1Ac in their toxicity to Plutella xylostella larvae.     

Involvement of miR164- and miR167-mediated target gene expressions in responses to water deficit in cassava

Cassava (Manihot esculenta Crantz) is an important crop and it is significantly affected by water stress. The computational analysis of cis-regulatory elements in promoter regions of 21 drought-responsive miRNA gene families and 35 miRNA-target genes in cassava indicated some elements relevant to drought stress responses. To investigate the role of miRNAs and target genes in responses to a water deficit in cassava in more detail, in vitro plantlets were subjected to an imitated water deficit by 40 % polyethylene glycol. Using RT-qPCR, the differential expression of the cassava miR164/MesNAC and miR167/MesARF6/8 were observed to be associated with changes in the leaf shape, stomatal closure, and relative water content. The modified 5′-RNA ligase-mediated rapid amplification of cDNA-end (5’RLM-RACE) experiment confirmed MesNAC and MesARF8 as the in vivo-target genes of miR164 and miR167, respectively, in cassava leaf. The possible functions of miR164 and miR167-target genes in response to water deficit are discussed.     

Constructing Bacillus thuringiensis strain that co-expresses Cry2Aa and chitinase

A triple recombineering technique was used with plasmid pHT315 to produce pHTEC, a construct carrying chitinase and cry2Aa genes from Bacillus thuringiensis subsp. kurstaki 4.0718. Transformation of wild-type B. thuringiensis strain HD73 and the acrystalliferous strain Cry-B with pHTEC resulted in the recovery of recombinant strains that expressed Cry2Aa as cubic crystals in the cell pellet and soluble chitinase protein. The toxicity of HD73 (pHTEC) against Helicoverpa armigera larvae increased sevenfold when compared with HD73 (pHT315) harboring pHT315 vector. The triple recombineering protocol was optimized by comparing recombination efficacy mediated by RecE/RecT and Redα/Redβ and by using single-strand DNA as substrate.     

Estradiol inducible and flower-specific expression of ARGOS and ARGOS-LIKE genes in transgenic tobacco plants

Transgenic tobacco plants expressing Arabidopsis thaliana ARGOS and ARGOS-LIKE genes under the control of the chalcone synthase promoter of Petunia hybrida L., as well as the estradiol inducible XVE system, have been obtained. The part of transgenic plants with flower-specific expression of the target genes was characterized by increased flower size, caused by an increase in cell size and quantity in the case of the ARGOS gene and by a stimulation of cell growth via stretching in the case of the ARGOS-LIKE gene. An enhanced expression level of the NtEXPA1, NtEXPA4 genes encoding expansins, NtEXGT gene encoding endo-xyloglucan transferase, and the AINTEGUMENTA-like gene was detected in the flowers of transgenic tobacco plants. In the case of inducible expression of ARGOS and ARGOS-LIKE genes, an increase in leaf, stem and flower size was revealed in several lines of transgenic plants as compared to control. Expression of the ARGOS gene also affected cell number and size in this case, while the ARGOS-LIKE gene mainly influenced cell size via stretching. Inducible expression of the ARGOS gene in flowers mainly provided an enhanced containment of AINTEGUMENTA-like mRNA, while ARGOS-LIKE gene expression resulted in the activation of NtEXPA1 and NtEXGT genes.     

Transgenic rice expressing the <Emphasis Type="Italic">cry</Emphasis>2AX1 gene confers resistance to multiple lepidopteran pests

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34 µg g^?1 leaf fresh weight and with 80–92 % levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7–3.6 ng g^?1). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

Expression status of let-7a and miR-335 among breast tumors in patients with and without germ-line BRCA mutations

The genetic factors of cancer predisposition remain elusive in the majority of familial and/or early-onset cases of breast cancer (BC). This type of BC is promoted by germ-line mutations that inactivate BRCA1 or BRCA2. On the other hand, recent studies have indicated that alterations in the levels of miRNA expression are linked to this disease. Although BRCA1 and BRCA2 gene mutations have been reported to commonly lead to alterations in genes that encode cancer-related proteins, little is known regarding the putative impact of these mutations on noncoding miRNAs. In the present study, we aimed to determine whether miRNA dysregulation is involved in the pathogenesis of BRCA-mutated BC. An expression analysis of 14 human miRNAs previously shown to be related to BC diagnosis, prognosis, and drug resistance was conducted using tissues from 60 familial and/or early-onset patients whose peripheral blood samples had been screened for BRCA1 and BRCA2 mutations through sequence analysis. Let-7a and miR-335 expression levels were significantly downregulated in the tumors of patients with a BRCA mutation compared with those of patients without a BRCA mutation (P = 0.04 and P = 0.02, respectively). Our results defined the associations between the expression status of let-7a and miR-335 and BRCA mutations. The expression analysis of these miRNAs might be used as biomarkers of the BRCA mutation status of early-onset and/or familial BC.     

NDRG1 expression is related to the progression and prognosis of gastric cancer patients through modulating proliferation,invasion and cell cycle of gastric cancer cells

N-myc downstream-regulated gene 1 (NDRG1) has been proposed as a tumor suppressor gene in many different types of tumors, but its potential function and corresponding mechanism are not yet fully elucidated. This study aims to detect the possible function of NDRG1 in gastric cancer progression. In this study, 112 paired gastric cancer tissues and corresponding nonmalignant gastric tissues were utilized to identify the differential protein expression of NDRG1 by immunohistochemistry and its clinical significance was analyzed. Furthermore, 49 of 112 paired gastric specimens were used to detect the differential mRNA expression by real-time PCR. The over expression of NDRG1 in human gastric cancer cell line AGS by PcDNA3.1–NDRG1 transfection was utilized to detect the role of NDRG1 in regulating the biological behavior of gastric cancer. NDRG1 expression was significantly decreased in primary gastric cancer tissues, compared with its corresponding nonmalignant gastric tissues (p < 0.05), and its decreased expression was significantly associated with lymph node metastasis (p < 0.01), invasion depth (p < 0.01) and differentiation (p < 0.05). Additionally, the overall survival rate of gastric cancer patients with high expression of NDRG1 was higher than those with low expression during the follow-up period. NDRG1 overexpression suppressed cells proliferation, invasion and induced a G1 cell cycle arrest in gastric cancer. Furthermore, the down-regulation of NDRG1 in gastric cancer metastatic progression was correlated to E-cadherin and MMP-9. Our results verify that NDRG1 acts as a tumor suppressor gene and may play an important role in the metastasis progression and prognosis of gastric cancer.     

Development of leaffolder resistant transgenic rice expressing <Emphasis Type="Italic">cry2AX1</Emphasis> gene driven by green tissue-specific <Emphasis Type="Italic">rbcS</Emphasis> promoter

The insecticidal cry genes of Bacillus thuringiensis (Bt) have been successfully used for development of insect resistant transgenic rice plants. In this study, a novel cry2AX1 gene consisting a sequence of cry2Aa and cry2Ac gene driven by rice rbcS promoter was introduced into a rice cultivar, ASD16. Among 27 putative rice transformants, 20 plants were found to be positive for cry2AX1 gene. The expression of Cry2AX1 protein in transgenic rice plants ranged from 5.95 to 122.40 ng/g of fresh leaf tissue. Stable integration of the transgene was confirmed in putative transformants of rice by Southern blot hybridization analysis. Insect bioassay on T₀ transgenic rice plants against rice leaffolder (Cnaphalocrosis medinalis) recorded larval mortality up to 83.33 %. Stable inheritance and expression of cry2AX1 gene in T₁ progenies was demonstrated using Southern and ELISA. The detached leaf bit bioassay with selected T₁ plants showed 83.33–90.00 % mortality against C. medinalis. The whole plant bioassay for T₁ plants with rice leaffolder showed significant level of resistance even at a lower level of Cry2AX1 expression varying from 131 to 158 ng/g fresh leaf tissue during tillering stage.     

Expression analysis and functional characterization of a cold-responsive gene COR15A from Arabidopsis thaliana

The cold-responsive (COR) genes play an important role in cold acclimation of higher plants. Here, a tight correlation between chloroplast functionality and COR15A expression, and the functional characterization of Arabidopsis COR15A involved in salt/osmotic stress, were revealed. COR15A gene is light inducible and expressed in light-grown seedlings. The expression level of COR15A was reduced when chloroplasts were damaged by norflurazon treatment. By using several albino mutants, seca1, secy1, and tic20, all of which exhibited severe defects in both structure and function of chloroplast, it was shown that the accumulation of COR15A mRNA depends on chloroplast functionality. Real-time RT-PCR and GUS-staining assays demonstrated that COR15A was induced by salt/osmotic stress partially via ABA. Overexpression of COR15A in Arabidopsis resulted in the seedlings displaying hypersensitivity to salt/osmotic stress. All these results suggest that plant acquire the ability to fully express COR15A only after the development of functional chloroplasts, COR15A may be involved in response to salt/osmotic stress during early stages of plant development.