An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

Regulation of AtbZIP1 Gene Expression by T-DNA Insertion in Arabidopsis thaliana

bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mutant Arabidopsis （bzipl） were used with T-DNA inserted into two different sites, designated as SALK-556773 and SALK-660942, in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites. PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression, while T-DNA insertion in 3＇-UTR couldn＇t. The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants＇ drought resistance, while in 3＇-UTR couldn＇t. The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.

The Effect of Windings on ADSL Transformer Insertion Losses

Insertion loss （IL） is one of the important parameters of asymmetrical digital subscriber loop （ADSL） transformers. In different frequency bands, the factors that affect insertion loss are different. Windings mainly affect insertion loss in mid and high frequency bands. The effects of winding ways, winding wire diameter and winding turns on insertion loss were discussed. The presented experiment shows that the insertion loss of an ADSL transformer could be under 0.4 dB in mid frequency band when the winding is 30 turns, in which the ADSL transformer satisfies the requirement of total harmonic distortion （THD）. Our experiments also show that the sandwich winding structure is better than the side by side winding structure and the twisted-pair winding structure, and the increase of winding diameter is one means to reduce insertion losses of an ADSL transformer in mid frequency band.

Agrobacterium-Mediated Transformation of Rice: Constraints and Possible Solutions

Genetic transformation of rice(Oryza sativa L.) by introducing beneficial traits is now a central research instrument in plant physiology and a practical tool for plant improvement. Many approaches are verified for stable introduction of foreign genes into the plant genome. The review examined the different constraints that limit the success of rice genetic transformation via Agrobacterium-mediated approach and suggested possible solutions. Explant identification, gene transfer technique and construct to tailor the integration, transgene expression without collateral to genetic damage and transformant selection are among the technical challenges affecting the rice transformation. Despite the contests, Agrobacteriummediated transformation system has been a better option for producing transgenic rice varieties because of its exact T-DNA processing and simple integration of low copy-number transgene. This information is necessary for improving the transformation system for recalcitrant rice varieties.

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

Identification of T-DNA Inserted Mutant Gene Transcription by Using Real-time PCR in Arabidopsis

AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （ABA） and ethylene stimulus ATSYR1 gene encodes a syntaxin localizing at the plasma membrane in Arabidopsis, which can be induced by abiotic stress. To identify mutation lines for gene functional analysis, real-time PCR was employed to detect the expression level of AtERF4 and ATSYR1 in homozygous T-DNA insertion mutant line, respectively. Real-time PCR is a powerful tool which can be used to detect steady-state mRNA levels specifically, sensitively and reproducibly. Comparing to other forms of quantitative RT-PCR, the amount of amplified products can be detected by real-time PCR instantly and thus is a preferable alternative. In this study, RNA with T-DNA inserting into exon could be detected in AtERF4 knock-out mutation line. The results indicated that AtERF4 had been trucked in transcription level. On the other hand, T-DNA inserting into the promoter of gene ATSYR1 had no effect on reducing the expression level ofATSYR1 gene. Further molecular and phenotype studies now are ongoing to clarify the potential consequences of AtERF4 and ATSYR1 deficiency in Arabidopsis

Optimizing Culture System of Ri T-DNA Transformed Roots for Citrus grandis cv. Changshou Shatian You

Genetic transformation experiments of the different explants from Citrus grandis cv. Changshou Shatian You infected with Agrobacterium rhizogenes were carried out in darkness or in light. The optimizing culture system of Ri T-DNA transformed roots for C. grandis cv. Changshou Shatian You was constructed as follows： After the ventral wounded striations on the single activation cotyledon were inoculated by A. rhizogenes A4 （logarithmic period）, they were cocultured at （25 ±2）℃ in darkness for 25-30 days; some transformed roots were generated from wounded striations of most cotyledons. The genetically transformed ratio is （83 ± 11）%. Axenic Ri T-DNA transformed roots （hairy roots） were harvested after five subcultures. Explants were activated on MT medium. The MS medium was used for subculture of transformed roots. Mass Ri T-DNA transformed roots in which the hormone was produced independently were harvested from this optimizing culture system. White, fresh Ri T-DNA transformed roots were （1.14 ±0.07） cm long, （0.73 ±0.04） mm wide, and the growth direction of transformed roots was negative geotropism.

T-DNA插入产生的水稻小粒突变体的遗传分析(英文)

[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-DNA tag lines, the progeny of homozygous plants carrying T-DNA insertion were screened for mutants with mutated phenotypes. The genetic analysis of the mutant and test for the linkage between the mutated phenotype and the T-DNA insertion were carried out to determine its genetic characteristics. [Result] In the present study, a grain shape mutant induced by T-DNA insertion in rice was identified, which showed small grain. Genetic analysis of the mutant showed that the two types of phenotype, normal and small grain in the segregating populations derived from the T-DNA heterozygotes, fit the ratio of 3∶1. Test for Basta resistance showed that all the mutants were resistant while the normal plants segregated for resistant and susceptible by the ratio of 2∶1. The results indicated that the mutant phenotype cosegregated with Bar gene. The small grain mutant caused by T-DNA insertion was confirmed by PCR amplification aiming at T-DNA. [Conclusion] The grain shape mutant is useful for isolation of the tagged gene and genetic improvement in rice.

不同大豆基因型对大豆遗传转化效率的影响及外源T-DNA插入分析

大豆通常被认为是较难转化的豆科作物之一,大豆基因型是影响大豆转化效率的重要因素。采用农杆菌介导大豆子叶节遗传转化方法,对2个国外大豆品种和9个国内品种进行转化研究,对转化再生植株进行BAR试纸条和PCR检测。结果表明：不同大豆品种再生率及转化效率存在显著差异。国外品种Jack和Bert,南方大豆品种华春6号和东北大豆品种沈农9号具有较高的再生率和转化效率,转化效率分别达到6.45%、3.80%、3.24%和2.86%。对来源于沈农9号的PCR阳性植株进行Southern杂交检测,结果表明：外源基因已导入受体大豆品种,该受体品种实际转化效率为2.14%。对外源T-DNA插入结构进一步分析表明：低拷贝（1-2个）T-DNA插入比例为75%。本研究筛选了4个转化效率较高的大豆品种,为开展高效大豆遗传转化研究提供依据;同时,作为大豆主栽品种,利用华春6号和沈农9号作为受体品种开展转基因研究,对于加快转基因大豆新品种培育研究也具有重要的参考价值。

T-DNA插入麝香百合的研究(英文)

采用2步外植体法结合比较成熟的农杆菌介导T—DNA插入技术转化麝香百合，为快速选育百合新品种奠定基础。

T-DNA插入麝香百合的研究

[目的]获得麝香百合转基因植株。[方法]采用2步外植体法和农杆菌介导的T-DNA插入技术转化麝香百合。[结果]菌液浓度OD600值为0．6～0．8时浸染效果好，同时附加250mg／L的AS可以提高转化效率，50mg／L G418为抗性筛选最适浓度。对经过抗性筛选的百合转化植株进行PCR分子检测，部分转基因植株呈阳性，初步证明T-DNA已插入到百合基因组中。[结论]该研究为利用T-DNA插入技术筛选优良的百合新品种奠定了基础。

棉花黄萎病菌T-DNA插入突变体库的构建及致病缺陷突变体筛选

大丽轮枝菌(Verticillium dahliae)侵染引起的棉花黄萎病是棉花生产上的重要病害之一,而其致病的分子机制尚不清楚,为此,以强致病力落叶型黄萎病菌FGH2为材料,利用农杆菌介导转化技术构建棉花黄萎病菌T-DNA插入突变体库并进行致病缺陷突变体的筛选。当农杆菌与黄萎病菌分生孢子浓度比为250∶1、承载介质为NC膜、乙酰丁香酮浓度400μmol/L、共培养时间48h时,转化效率高达每106个分生孢子产生612.5个转化子。通过采用优化后的转化体系,构建了含1 200个T-DNA插入转化子的棉花黄萎病菌突变体库。利用苗期分生孢子蘸根法测定300个转化子在棉花幼苗上的致病力,获得3个致病力显著降低的突变体181、546、1009,其中546的微菌核形成能力丧失,1009的微菌核形成能力明显下降。黄萎病菌T-DNA插入突变体库的构建,为棉花黄萎病菌致病及微菌核形成机制的研究奠定了基础。

稻曲病菌T-DNA插入突变体B2510的插入位点分析

以稻曲病菌T-DNA插入突变体库中致病力减弱突变菌株B2510为材料,通过分析T-DNA插入位点的侧翼序列和突变基因,分离出在稻曲病菌致病过程中起作用的基因。通过测定突变菌株B2510的生长速率、产孢能力及致病力发现,与野生型菌株P1相比,B2510田间接种表现为致病性减弱;在MM培养基上生长速率下降,而在PSA和TB3培养基中生长速率与野生型没有显著差异,但丧失产孢能力。Southern杂交显示T-DNA在突变菌株B2510中以双拷贝形式插入,利用TAIL-PCR技术扩增紧邻T-DNA两侧的侧翼序列,经过比对分析发现,T-DNA分别插在基因UV8b_1412的启动子区域和UV8b_1386的下游3′端,且稻曲菌基因组序列均未丢失,T-DNA上只有几个碱基发生变化。半定量RT-PCR分析基因的表达情况,显示两个基因在突变体B2510的表达量较P1均显著下降,推测T-DNA插入位点处的基因与稻曲病菌致病性相关,可能在某一阶段参与调控稻曲病菌在水稻上的致病过程。

油菜黑胫病菌T-DNA插入诱变因素优化及突变体筛选

油菜黑胫病是由Leptosphaeria biglobosa引起的一种真菌病害。这种病害在我国油菜产区广泛发生,并造成一定的经济损失。为通过获取突变体来研究L.biglobosa的生态适应性机制及致病机制,本文优化了影响农杆菌介导转化(ATMT)油菜黑胫病菌L.biglobosa菌株Lb731的因素,评估转化子质量,并筛选相关突变体。结果明确了农杆菌介导转化菌株Lb731的最佳因素:潮霉素B浓度为50μg/mL,转化受体(分生孢子)培养时间为15 d(20℃),浓度为2×10^(7-8)孢子/mL,农杆菌-受体共培养温度为25℃,共培养时间为72 h。在最适条件下的转化效率达到80个转化子/百万分生孢子。T-DNA插入基因组的频率为100%,单拷贝插入频率为72.7%,转化子抗潮霉素性状能稳定遗传。从2136个转化子中获得了11个菌丝生长减缓突变体,7个色素产生缺陷突变体和14个分生孢子产生缺陷突变体,并从这些突变体中鉴定出7个致病力丧失突变体。采用hiTAIL-PCR技术,从3个突变体中获得了T-DNA插入位点侧翼序列。上述结果为深入研究L.biglobosa的生态适应性机制及致病机制提供了材料和线索。

用T-DNA插入突变的方法获得稻瘟病菌致病突变体

利用农杆菌介导转化的方法共得到1 114个稻瘟病菌转化子,经感病大麦和水稻离体叶片接种鉴定获得3个致病缺陷或下降突变体:A1-134、A1-452、和A2-1-8,其中A1-134和A1-452的致病性下降,而A2-1-8突变体的致病性丧失。对这些突变体的分生孢子形态、产孢、附着胞形成等表型分析的结果发现,A1-134突变体的产孢量显著下降,仅为野生菌株Guy11的7.7%,分生孢子为圆球形,无隔膜或只有一个隔膜;A1-452产孢量明显增多,为Guy11的22倍;A2-1-8的分生孢子为长针形,产孢量略有下降;A1-134和A1-452在疏水表面的附着胞形成率与Guy11相近,而A2-1-8则不能形成附着胞。研究结果为进一步鉴定和分离这些突变体的T-DNA标记基因奠定了基础。

希金斯刺盘孢T-DNA插入突变体表型筛选及其特性分析

以希金斯刺盘孢菌株Ch-1为供试野生型菌株,通过农杆菌介导转化方法获得含2000个转化子的T-DNA插入突变体库,筛选菌落生长异常或致病缺陷突变体,分析致病缺陷突变体的T-DNA插入拷贝数和位点。结果显示,筛选到14株菌落异常突变体,包括2株生长缓慢突变体Ch-1-E393和Ch-1-C135、12株菌落形态异常突变体(包括色素异常、菌落扇变或菌丝坍塌现象),另外筛选到1株致病缺陷突变体Ch-1-G090。致病性测定结果表明,致病缺陷突变体Ch-1-G090接种拟南芥后,叶片发病明显减弱,且未产生水渍状病斑。显微观察发现该突变体分生孢子在叶片上仅能产生少量初生菌丝,不能正常形成次生菌丝。Southern杂交显示,致病缺陷突变体Ch-1-G090为T-DNA双拷贝插入;通过Inverse-PCR法获得插入T-DNA的侧翼序列,明确该突变体T-DNA插入位点分别为假定蛋白(Ch063_10682)和RNA加工蛋白FCF1(CH063_10671)编码区。

Effort and Contribution of T-DNA Insertion Mutant Library for Rice Functional Genomics Research in China:Review and Perspective

With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

The alteration in the architecture of a T-DNA insertion rice mutant osmtd1 is caused by up-regulation of Micro RNA156f

Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter-Binding Protein-Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T-DNA insertion mutant Osmtd1 (Oryza sativa multi-tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T-DNA insertion in Osmtd1. Further analysis revealed that the T-DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild-type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.

Induction of the AOX1D Isoform of Alternative Oxidase in A. thaliana T-DNA Insertion Lines Lacking Isoform AOX1A Is Insufficient to Optimize Photosynthesis when Treated with Antimycin A

Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome pathway （CP） that is linked to ATP production, and the alternative pathway （AP）, where electrons from ubiquinol are directly transferred to O2 via an alternative oxidase （AOX） without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T- DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOXIA expression in the wild-type, led to an increased expression of AOXID in leaves of the aoxla-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn- thesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild- type, which was only marginally affected by the inhibitor. It thus appears that AOX1 D was unable to fully compensate for the loss of AOXIA when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex （GDC）, suggests that the aoxla mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOXIA to optimize pho- tosynthesis when treated with antimycin A. We suggest that the aoxla mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

脱插机构的误差灵敏度分析与优化设计

本文通过建立发射箱脱插机构的几何误差传递模型,系统分析各组成零部件的加工装配误差对脱插机构空间误差灵敏度系数,确定脱插机构下连杆座与脱插机构基座之间、下连杆座与平动杆之间以及平动杆与上连杆座之间沿y轴方向的转动误差为关键误差来源,针对性地提出了改进措施并加以实施。最后,通过试验验证了误差灵敏度分析的有效性和改进措施的正确性。该方法为整个系统的误差优化分析提供了可靠的理论支撑,大幅度提高了脱插机构对接成功率。