An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton

The HUB2 gene encoding histone H2B monoubiquitination E3 ligase is involved in seed dormancy, flowering timing, defence response and salt stress regulation in Arabidopsis thaliana. In this study, we used the cauliflower mosaic virus (CaMV) 35S promoter to drive AtHUB2 overexpression in cotton and found that it can significantly improve the agricultural traits of transgenic cotton plants under drought stress conditions, including increasing the fruit branch number, boll number, and boll‐setting rate and decreasing the boll abscission rate. In addition, survival and soluble sugar, proline and leaf relative water contents were increased in transgenic cotton plants after drought stress treatment. In contrast, RNAi knockdown of GhHUB2 genes reduced the drought resistance of transgenic cotton plants. AtHUB2 overexpression increased the global H2B monoubiquitination (H2Bub1) level through a direct interaction with GhH2B1 and up‐regulated the expression of drought‐related genes in transgenic cotton plants. Furthermore, we found a significant increase in H3K4me3 at the DREB locus in transgenic cotton, although no change in H3K4me3 was identified at the global level. These results demonstrated that AtHUB2 overexpression changed H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus, leading the GhDREB gene to respond quickly to drought stress to improve transgenic cotton drought resistance, but had no influence on transgenic cotton development under normal growth conditions. Our findings also provide a useful route for breeding drought‐resistant transgenic plants.     

Transgenic cotton expressing synthesized scorpion insect toxin AaHIT gene confers enhanced resistance to cotton bollworm (Heliothis armigera) larvae

A synthetic scorpion Hector Insect Toxin (AaHIT) gene, under the control of a CaMV35S promoter, was cloned into cotton via Agrobacterium tumefaciens-mediated transformation. Southern blot analyses indicated that integration of the transgene varied from one to more than three estimated copies per genome; seven homozygous transgenic lines with one copy of the T-DNA insert were then selected by PCR and Southern blot analysis. AaHIT expression was from 0.02 to 0.43% of total soluble protein determined by western blot. These homozygous transgenic lines killed larvae of cotton bollworm (Heliothis armigera) by 44–98%. The AaHIT gene could used therefore an alternative to Bt toxin and proteinase inhibitor genes for producing transgenic cotton crops with effective control of bollworm.     

陆地棉GhCDPK1基因响应干旱胁迫的功能初探

钙依赖性蛋白激酶(CDPKs)是一类重要的钙信号感受蛋白和响应蛋白,在植物干旱、低温、盐碱等非生物胁迫应答中起着重要的调控作用。为探讨陆地棉GhCDPK1基因在干旱胁迫下所起的作用,该研究利用实时荧光定量PCR技术分析了PEG模拟干旱胁迫下该基因的表达量,发现GhCDPK1基因受干旱胁迫诱导。通过构建植物表达载体pCAMBIA2300-GhCDPK1,采用农杆菌介导的叶盘法转化模式植物烟草,发现干旱胁迫下转基因植株保水能力明显高于野生型植株,叶绿素、脯氨酸、可溶性蛋白含量及POD、SOD活性也高于野生型植株,而丙二醛含量低于野生型植株。研究结果表明,GhCDPK1基因作为正向调控因子响应干旱胁迫诱导,过表达GhCDPK1基因可以使植株积累更多的渗透调节物质、增强抗氧化系统酶的活性和维持细胞膜的稳定性来提高植物抵御外界干旱胁迫的能力。     

Transgenic Agrostis mongolica Roshev. with enhanced tolerance to drought and heat stresses obtained from Agrobacterium-mediated transformation

Efficient procedures for regeneration and Agrobacterium-mediated transformation were established for Agrostis mongolica Roshev. and generated transgenic plants tolerant to drought and heat stresses using a regulatory gene from Arabidopsis, ABF3, which controls the ABA-dependent adaptive responses. The identification and selection of regenerable and reproducible callus type was a key factor for successful transformation. The transformation efficiency was 49.2% and gfp expression was detected in hygromycin-resistant calli and stem of putative transgenic plants. The result of Southern blot analysis showed that the ABF3 transgene was stably integrated into the genome of transgenic plants. Of the five transgenic lines analyzed, single transgene integration was observed in two lines and two copy integration was observed in three transgenic lines. Northern blot analysis confirmed that ubi::ABF3 was expressed in all transgenic lines. Transgenic plants exhibited neither growth inhibition nor visible vegetative phenotypic alternations. However, both transgenic and wild-type plants were highly sterile and did not flower during 3 years of growth period in the open field under subtropical Jeju Island climate. The stomata of the transgenic plants opened less than did stomata of the wild-type plants, and water content of the transgenic leaves remained about 3–4 fold higher than observed for wild-type leaves under drought stress. The transgenic plants showed about 2 fold higher survival rates under drought stress and about 3 fold higher survival rates under heat stress when compared to wild-type plants. Thus, overexpression of the Arabidopsis ABF3 gene results in enhancement of both drought and heat stress tolerance in Agrostis mongolica Roshev.     

Introduction and constitutive expression of a tobacco hornworm (<Emphasis Type="Italic">Manduca sexta</Emphasis>) chitinase gene in soybean

Summary Embryogenic soybean [Glycine max (L.) Merrill] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration inheritance, and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes. Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of 48kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode.     

转大肠杆菌过氧化氢酶基因棉花的抗旱生理研究

以导入大肠杆菌过氧化氢酶基因KatE的T3代转基因棉花为供试材料,经卡那霉素检测和PCR鉴定,将筛选出的阳性转基因植株与对照棉花进行整个生育期的持续水分胁迫处理直至收获,比较材料间的生理生化指标的差异,鉴定转基因植株的耐旱能力。结果显示:(1)干旱胁迫持续至初蕾期时,转基因棉花与对照植株间各项抗旱生理指标差异均未达到显著水平。(2)水分胁迫持续至盛蕾和盛花期时,转基因棉花叶片相对含水量、光系统Ⅱ最大光化学效率(Fv/Fm)、CAT活性,以及叶片的净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均显著或极显著高于对照植株,叶绿素含量也都明显高于对照植株。干旱胁迫持续至吐絮期时,转基因棉花的株高、果枝数和铃数均显著或极显著高于对照植株,且转基因棉花和对照的籽棉产量分别比正常灌溉处理降低57.5%和60.1%,全生育期的水分胁迫严重影响了棉花籽棉产量,但转基因棉花的籽棉产量仍显著高于对照。研究表明,在新疆石河子当地自然降水(干旱胁迫)条件下,转KatE基因棉花表现出了较好的生理和生长优势,KatE基因有助于提高棉花的抗旱性。     

Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis

Fusarium head blight (scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat (Triticum aestivum L.) worldwide. Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the major diseases of wheat in China. The defensin RsAFP2, a small cyteine-rich antifungal protein from radish (Raphanus sativus), was shown to inhibit growth in vitro of agronomically important fungal pathogens, such as F. graminearum and R. cerealis. The RsAFP2 gene was transformed into Chinese wheat variety Yangmai 12 via biolistic bombardment to assess the effectiveness of the defensin in protecting wheat from the fungal pathogens in multiple locations and years. The genomic PCR and Southern blot analyses indicated that RsAFP2 was integrated into the genomes of the transgenic wheat lines and heritable. RT-PCR and Western blot proved that the RsAFP2 was expressed in these transgenic wheat lines. Disease tests showed that four RsAFP2 transgenic lines (RA1–RA4) displayed enhanced resistance to F. graminearum compared to the untransformed Yangmai 12 and the null-segregated plants. Assays on Q-RT-PCR and disease severity showed that the express level of RsAFP2 was associated with the enhanced resistance degree. Two of these transgenic lines (RA1 and RA2) also exhibited enhanced resistance to R. cerealis. These results indicated that the expression of RsAFP2 conferred increased resistance to F. graminearum and R. cerealis in transgenic wheat.     

A novel wheat α‐amylase inhibitor gene,TaHPS, significantly improves the salt and drought tolerance of transgenic Arabidopsis

On the basis of microarray analyses of the salt‐tolerant wheat mutant RH8706‐49, a previously unreported salt‐induced gene, designated as TaHPS [Triticum aestivum hypothetical (HPS)‐like protein], was cloned. Real‐time quantitative polymerase chain reaction analyses showed that expression of the gene was induced by abscisic acid, salt and drought. The encoded protein was found to be localized mainly in the plasma membranes. Transgenic Arabidopsis plants overexpressing TaHPS were more tolerant to salt and drought stresses than non‐transgenic wild‐type (WT) plants. Under salt stress, the root cells of the transgenic plants secreted more Na⁺ and guard cells took up more Ca²⁺ ions. Compared with wild‐type plants, TaHPS‐expressing transgenic plants showed significantly lower amylase activity and glucose and malic acid levels. Our results showed that the expression of TaHPS inhibited amylase activity, which subsequently led to a closure of stomatal apertures and thus improved plant tolerance to salt and drought.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated co-transformation of multiple genes in upland cotton

Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis of T₁ plants demonstrated that multiple transgenes were inherited and expressed in progenies. Fei-Fei Li and Shen-Jie Wu are joint first authors.     

转GhDREB小麦株系‘G鲁麦23’抗旱性分析

以T6代转抗旱相关基因GhDREB小麦株系‘G鲁麦23’及其受体对照‘鲁麦23’为材料,通过PEG-6000人工模拟干旱胁迫处理观察小麦萌发期胚芽鞘长度、胚根数、胚根长度,采用大田不同水分处理试验研究其不同生育时期的生理生化、形态指标与抗旱指数,以明确转基因材料的抗旱性.结果表明:(1)在种子萌发期20%(W/V)PEG-6000干旱胁迫条件下,‘G鲁麦23’及其受体对照‘鲁麦23’的胚芽鞘长度、胚根长度和胚根数均比非胁迫时降低,且‘G鲁麦23’的降幅较小但均显著高于受体对照;(2)与对照鲁麦23相比,‘G鲁麦23’具有较高的叶片相对含水量和可溶性蛋白含量;(3)随干旱胁迫程度的增强‘G鲁麦23’与‘鲁麦23’株高均有不同程度的降低,但G鲁麦23株高始终高于‘鲁麦23’;在不浇水条件下,‘G鲁麦23’穗叶距显著长于‘鲁麦23’;而3种水分处理下‘G鲁麦23’与‘鲁麦23’穗长几乎没有变化;(4)‘G鲁麦23’在不浇水和浇1次水条件下分别比对照增产11.68%和9.05%,其抗旱指数为1.22,属较强抗旱等级,表现出比‘鲁麦23’更强的抗旱、节水性能.     

High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2, by constitutive and inducible expression of tobacco <Emphasis Type="Italic">osmotin</Emphasis>

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.     

转OvDREB2B基因陆地棉鉴定及其对水分渗透胁迫的分析

通过花粉管通道技术,以该实验室自育陆地棉品系TH₁和TH₂为材料,将诸葛菜(Orychophragmus vidaceus)抗逆转录因子OvDREB2B基因构建到植物表达载体后,导入棉花基因组,经卡那霉素筛选和分子鉴定表明目的基因已整合到棉花基因组中并表达。将T₁代转基因植株和受体对照在温室中栽培,待植株生长至四叶一心时,用不同渗透势的PEG-6000水溶液进行渗透胁迫处理,分析探讨转基因植株的抗旱效果及其抗旱机理。结果显示:当渗透势为0和0.5 MPa处理时,转基因植株和对照无明显差异;当渗透势为0.8 MPa和1.1 MPa处理时转基因植株较对照抗旱性明显提高。当渗透势为1.1 MPa处理96 h时,对照植株F_v/F_m降至0.2左右,而转基因植株仍正常生长,F_v/F_m值约为0.51,而且初始荧光（F₀）值、净光合速率（P_n）、胞间CO₂浓度（C_i）、蒸腾速率（T_r）等一系列参数转基因植株都明显优于对照,表明DREB2B基因能够提高棉花对水分胁迫的耐受性。     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

A Novel ABA-Responsive TaSRHP Gene from Wheat Contributes to Enhanced Resistance to Salt Stress in Arabidopsis thaliana

A microarray analysis of the salt-resistant wheat mutant, RH8706-49, revealed a salt-induced gene containing a conserved DUF581 domain. The gene was cloned and designated as Triticum aestivum salt-related hypothetical protein (TaSRHP) and submitted to GenBank (accession no. GQ476575). Over-expression of TaSRHP in wild-type Arabidopsis thaliana cv. Columbia resulted in enhanced resistance to both salt and drought stresses. The sensitivity of the transgenic A. thaliana to abscisic acid (ABA) was also increased compared to that of wild-type plants. Furthermore, transgenic plants accumulated more K⁺ and proline and had a higher osmotic potential and lower Na⁺ content than untransformed plants. Real-time quantitative PCR analysis indicated that expression of TaSRHP was affected by salt, drought, cold, ABA, and other stresses, and expression of other stress-related genes in the transgenic plants differed from those of the control. Results indicate that the wheat TaSRHP gene may enhance the tolerance of plants to multiple abiotic stresses.     

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) and Regeneration of Salt Tolerant Plants

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (km^r) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T₁, T₂, and T₃ progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T₁ AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.     

Enhanced drought and salinity tolerance in transgenic potato plants with a BADH gene from spinach

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = −2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4–0.9 cm and fresh weight per plant increased by 17–29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.