根癌农杆菌介导的日本曲霉转化体系的建立

【目的】通过根癌农杆菌介导的方法构建日本曲霉转化子库,从而筛选出高产甘没氧化酶的日本曲霉突变菌株。【方法】本文通过三亲杂交的方法将双元载体pBI-hphII转移至根癌农杆菌EHA105中并作为侵染菌株,以日本曲霉As5999为受体菌株,建立了农杆菌介导的日本曲霉转化体系,构建了突变体库,并对影响转化效率的根癌农杆菌浓度,乙酰丁香酮(As)加入与否,共培养时间,共培养温度等因素进行了分析。【结果】对转化子的PCR检测和Southern杂交分析表明,T-DNA已整合进日本曲霉基因组中,随机挑选的9个转化子连续转接10代后均能稳定遗传。【结论】该转化体系的建立为筛选出高产甘油氧化酶的日本曲霉突变菌株奠定了基础。     

根癌农杆菌介导的赭曲霉遗传转化体系的建立

赭曲霉在工业上用于甾体C-11α羟基化。为了对现有赭曲霉生产菌株进行遗传改造,以农杆菌LBA4404作为侵染菌株,以赭曲霉(TCCC41060)作为受体菌株,以潮霉素B基因作为筛选标记,成功建立了根癌农杆菌介导的赭曲霉遗传转化体系并对其转化效率的主要影响因素,如农杆菌浓度、孢子浓度、共培养时间和温度等进行了分析。在最佳条件下,转化效率可以达到57个转化子/108个分生孢子。随机挑选的8个阳性转化子10代连续培养结果表明所获得的转化子能稳定遗传。该遗传转化体系的建立为赭曲霉菌株的定向遗传改造提供了重要的操作平台。     

链格孢ATMT转化体系的构建及弱毒突变株验证

本文利用来自质粒pUCATPH的构巢曲霉色氨酸合成基因trpC的启动子(PtrpC)、终止子(Ttrpc)和潮霉素磷酸转移酶抗性基因(hph)以及植物表达载体pROK Ⅱ成功构建了适用于丝状真菌的根癌农杆菌介导转化的双元载体pROKIIHPH,并转化根癌农杆菌Agrobacterium tumefaciens LBA4404,建立了根癌农杆菌LBA4404介导的链格孢Alternaria alternata分生孢子转化体系;再从乙酰丁香酮(AS)浓度、不同的共培养时间、受体菌分生孢子浓度和农杆菌菌液浓度对A.alternata转化效率的影响对体系进行优化.结果确定了根癌农杆菌菌液体积为200 mL(OD_(600)=0.15),A.alternata分生孢子浓度为10~6个/mL,在A.tumefaciens的预培养时期以及与A.alternata共培养时期分别加入200 μmol/LAS,共培养时间48 h,转化率达120～200个/10~5分生孢子.在所筛选的约800个转化子中获得了1株毒性明显低于野生菌sd1的弱毒突变株t108,通过PCR验证推测其毒力降低可能由于T-DNA插入阻断了基因的表达.该实验结果为与突变相关基因的研究以及弱毒株t108的进一步研究和利用提供了实验材料,也为深入研究链格孢sd1菌株的基因功能奠定了基础.     

农杆菌介导的出芽短梗霉遗传转化及高效筛选聚苹果酸高产菌株

建立根癌农杆菌介导的出芽短梗霉遗传转化方法及T-DNA突变库,高效筛选聚苹果酸高产菌株及功能基因。通过含潮霉素和草铵磷抗性基因的农杆菌转化出芽短梗霉,抗性压力筛选及PCR验证建立根癌农杆菌介导的出芽短梗霉遗传转化方法,结合发酵液p H与聚苹果酸含量响应变化,微孔板高效筛选高产聚苹果酸的T-DNA插入突变株,基因组步移确定T-DNA插入位点及功能基因。结果获得遗传稳定的抗性基因菌株,每107个细胞可获得80-120个转化子,出芽短梗霉H27号T-DNA突变株聚苹果酸摇瓶发酵产量提高24.5%,基因组步移证实糖酵解途径磷酸甘油酸变位酶基因被破坏。成功建立了根癌农杆菌介导的出芽短梗霉遗传转化方法和T-DNA插入突变库,结合高效筛选方法为聚苹果酸合成功能基因挖掘及高产机制解析奠定基础。     

根癌农杆菌介导转化红曲菌及转化子发酵性能的研究

利用根癌农杆菌介导转化技术成功将潮霉素抗性基因转入发白红曲菌中,优化了抗生素浓度,发白红曲菌孢子浓度,根癌农杆菌浓度,共培养温度及时间,以及乙酰丁香酮浓度等转化条件,最终转化效率可达52个转化子/105个红曲孢子.将转化子在含有潮霉素B的培养基继代培养5代,得到了多株稳定的转化子,对部分转化子进行PCR鉴定,结果进一步...     

根癌农杆菌介导里氏木霉转化体系的建立和条件优化

目的:采用根癌农杆菌介导的转化方法实现丝状真菌里氏木霉的遗传转化,并优化转化条件.方法:构建含潮霉素抗性基因(hph)的双元载体pCAM-hph后,转化根癌农杆菌LBA4404获得转化菌株.将根癌农杆菌的转化菌株和里氏木霉的分生孢子共培养后在含100μg/mL潮霉素的抗性平板上筛选里氏木霉转化子,并采用PCR扩增和序列测定对转化子中的插入片段进行了分析.结果:使用根癌农杆菌介导的转化方法转化里氏木霉,每106个分生孢子可获得25.8个转化子.最佳的转化条件为:农杆菌初始浓度为OD660约为0.8,孢子数为106个,共培养时间为48h,pH为5.0～5.5,培养温度为28℃.结论:建立了根癌农杆菌介导的里氏木霉转化方法,并获得了最佳的转化条件.     

菰黑粉菌T-DNA突变体库的构建及分析

通过建立适用于菰黑粉菌Ustilago esculenta的农杆菌介导遗传转化(Agrobacterium tumefaciens-mediated transformation,ATMT)体系,构建菰黑粉菌T-DNA插入突变体库。针对性地筛选双核菌丝形成缺陷型转化子,并对T-DNA插入位点进行分析,为研究菰黑粉菌二态型转换的分子调控机理打下基础。以构建的菰黑粉菌自融合菌株TSP为出发菌株,以含有遗传霉素(G418)抗性基因(neo)的质粒为载体,通过ATMT构建菰黑粉菌T-DNA突变体库,并对诱导剂乙酰丁香酮(AS)浓度、转化的共培养时间、农杆菌浓度和菰黑粉菌芽孢子浓度等建库影响因素进行单因素条件试验,筛选最优条件;对继代培养的转化子基因组中的遗传霉素抗性基因进行PCR检测,验证转化子遗传稳定性;对突变体库中的转化子双核菌丝生长情况进行观察,测定其双核菌丝形成能力;对上述双核菌丝形成缺陷型转化子进行基因组重测序,分析其T-DNA插入位点。当遗传霉素浓度为75μg/mL时,菰黑粉菌的生长被完全抑制。当AS浓度为100μg/mL、共培养时间为24 h、孢子浓度为1×10⁵     

根癌农杆菌介导的黑曲霉遗传转化体系的建立及优化

基于根癌农杆菌介导的遗传转化方法的独特优点,研究黑曲霉转化过程中各主要影响因素,建立高效的黑曲霉遗传转化方法。构建双元载体pBI-hph,通过电转导入农杆菌LBA4404中,以黑曲霉TCCC41056为受体菌株,利用潮霉素B基因作为筛选标记,对影响转化效率的孢子悬液的新鲜程度及浓度、农杆菌菌液浓度、共培养时间、共培养温度这五个条件进行分析,建立根癌农杆菌介导的黑曲霉遗传转化体系。实验结果表明,上述条件对黑曲霉的转化效率有较大的影响,通过优化,黑曲霉转化效率可达83个转化子/10⁷分生孢子;整合到黑曲霉基因组的外源基因可以稳定遗传,在转接10代后遗传性能仍保持稳定,并在众多转化子中筛选得到了糖化酶活力提高18%的黑曲霉突变株。根癌农杆菌介导的黑曲霉转化体系的建立,为进一步研究黑曲霉的功能基因以及开发黑曲霉表达系统提供了有力的手段。     

根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T-DNA插入突变体的体系,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15-0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化率最高,可达60-120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。     

根癌农杆菌介导的轮枝镰孢菌遗传转化及T-DNA插入突变体的筛选

建立并优化了农杆菌介导转化轮枝镰孢菌Fusarium verticillioides获得T-DNA插入突变体的体系,在镰孢菌孢子浓度106个/mL、农杆菌OD600=0.15-0.20、乙酰丁香酮浓度为200μmol/mL的条件下共培养36h转化率最高,可达60-120个/106个孢子。共获得转化子1000多个,连续转接5代能够稳定遗传。PCR验证潮霉素B抗性基因已整合进转化子基因组DNA中,部分转化子表现为生长和形态异常。该转化体系的建立为研究该菌的致病机制和功能基因分析奠定了基础。     

红曲霉桔霉素的检测方法及红曲霉产桔霉素的判别方法*

建立了红曲霉真菌毒素桔霉素的HPLC检测方法。用谷氨酸和葡萄糖为唯一氮、碳源的培养基(MSG)液态摇瓶培养及红曲米固态培养,对30多株红曲霉产桔霉素的情况进行了普查,发现大多数红曲霉菌种可产生桔霉素。红曲霉的培养状态及条件对桔霉素的产生有重大影响。红曲霉菌种是否产桔霉素,可根据MSG培养基摇瓶发酵液中是否含有桔霉素来初步定性判断,为准确判断红曲霉菌是否产桔霉素,可采用多种培养法综合判断。     

Characteristic analysis of transformants in T-DNA mutation library of <Emphasis Type="Italic">Monascus ruber</Emphasis>

Monascus ruber, a red mold species, has been widely used in the fields of food and medicine. In this research, we transformed Monascus ruber spores using Agrobacterium tumefaciens as a tool for random insertional mutagenesis with the hygromycin phosphotransferase gene as the selected marker. Three types of mutants including citrinin-producing mutants, mutants with abnormal aerial hyphae and pigment change mutants were screened for molecular analysis. Southern blot analysis showed that more than 83.3% of transformants contained single T-DNA insertions. The genomic DNA segments of the transformants flanking the T-DNA could be amplified from their left borders with TAIL-PCR. Homologous comparison using the Blast tool showed that none of the isolated DNA sequences had any similarity to each other, suggesting that the T-DNA was randomly integrated into the fungal genome, which provided the hypothetical reason for the variant phenotypes of the transformants. The successful creation of transformants with a single T-DNA tag insertion may help us to clone functional genes related to the metabolism and differentiation of Monascus spp., which will greatly facilitate the molecular analysis of this important fungus and the improvement of strains at the genetic level.     

农杆菌介导的红曲菌T-DNA插入突变库的构建及色素突变子的性质分析

以农杆菌EHA105为介导,将带有潮霉素抗性基因和gus基因的T-DNA片段转化到红色红曲菌(Monascusruber)中。通过转化条件的优化,成功构建了含有5132个转化子的T-DNA插入突变库。根据菌落颜色与色素分泌情况筛选到50株色素突变子,聚合酶链式反应(PCR)表明上述突变子均有T-DNA片段插入。经5代的继代培养后,94%的突变子具有稳定性(潮霉素抗性)。对突变子产色素和桔霉素能力的分析表明其分泌次生代谢产物的能力发生了较大变化。本方法的建立,为进一步深入研究红曲菌的代谢调节和基因功能奠定了基础。     

Cloning of Insertion Site Flanking Sequence and Construction of Transfer DNA Insert Mutant Library in Stylosanthes Colletotrichum

Stylosanthes sp. is the most important forage legume in tropical areas worldwide. Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, is a globally severe disease in stylo production. Little progress has been made in anthracnose molecular pathogenesis research. In this study, Agrobacterium tumefaciens-mediated transformation was used to transform Stylosanthes colletotrichum strain CH008. The major factors of the genetic transformation system of S. colletotrichum were optimized as follows: A. tumefaciens’ AGL-1 concentration (OD₆₀₀), 0.8; concentration of Colletotrichum conidium, 1×10⁶ conidia/mL; acetosyringone concentration, 100 mmol/L; induction time, 6 h; co-culture temperature, 25°C; and co-culture time, 3 d. Thus, the transformation efficiency was increased to 300–400 transformants per 106 conidia. Based on the optimized system, a mutant library containing 4616 mutants was constructed, from which some mutants were randomly selected for analysis. Results show that the mutants were single copies that could be stably inherited. The growth rate, spore amount, spore germination rate, and appressorium formation rate in some mutants were significantly different from those in the wild-type strain. We then selected the most appropriate method for the preliminary screening and re-screening of each mutant’s pathogenic defects. We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity. Thermal asymmetric interlaced PCR was used to identify the transfer DNA (T-DNA) integration site in the mutant that was coded 2430, and a sequence of 476 bp was obtained. The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome. By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae ({"type":"entrez-protein","attrs":{"text":"XP_003719674.1","term_id":"389644084","term_text":"XP_003719674.1"}}XP_003719674.1).     

高产γ-氨基丁酸低产桔霉素红曲霉(Monascus ruber)突变子1047筛选与发酵特性研究

以红曲米中筛选到的红色红曲霉菌株G为原始菌株,通过农杆菌介导T-DNA插入突变技术,成功构建了含有1 483株红曲霉突变子的T-DNA插入突变库。用HPLC等方法从突变库中筛选出10株γ-氨基丁酸(GABA)产量稳定高于原始菌株G的突变子,薄层层析结合HPLC技术分析了10株突变子发酵液中桔霉素的含量;其中突变子1047的GABA产量较高,为1.169g/L,是原始菌株G(GABA产量0.472g/L)的247.67%,桔霉素含量稳定低于原始菌株G。以红曲霉菌株G和突变子1047为实验材料,通过5 L发酵罐发酵并定时取样,HPLC等方法精确分析发酵液各种活性物质的含量;结果显示,突变子1047生长速度稍快于原始菌株G;GABA、莫纳可林K(Monacolin K)、红曲色素色价分别为2.201 g/L、83.892 mg/L、21.984 U/mL,是原始菌株G的279.67%、108.01%和182.35%;而桔霉素产量为1.976 mg/L,是原始菌株G的41.71%。因此,利用TDNA插入的方法对红曲霉进行育种,能产生稳定的遗传变化,在红曲霉资源的保护和利用上有一定潜力。     

Binary transformation systems based on 'shooter' mutants of Agrobacterium tumefaciens: a simple,efficient and universal gene transfer technology that permits marker gene elimination

A simple transformation procedure with a positive selection scheme using the expression of the isopentenyl transferase ( ipt) gene of transfer DNA (T-DNA) 'shooter' mutants of Agrobacterium tumefaciens was elaborated. After comparing several 'shooter' mutants we found that particular strains frequently produced phenotypically normal shoots after co-culturing with tobacco leaf explants. Shoots selected for normal phenotype showed apical dominance and could be rooted with the same efficiency as non-transformed shoots. When binary vectors were introduced into these strains, stably integrated binary vector T-DNA sequences were found in some regenerants, which were produced under non-selective conditions on growth-regulator-free medium. Such phenotypically normal transformants typically lacked a stably integrated ipt gene. Normal looking shoots could also be produced in tomato, muskmelon and sweet pepper.