Development of an Agrobacterium-mediated CRISPR/Cas9 gene editing system in jute(Corchorus capsularis)

Jute(Corchorus capsularis L.)is the second most important natural plant fiber source after cotton.However,developing an efficient gene editing system for jute remains a challenge.In this study,the transgenic hairy root system mediated by Agrobacterium rhizogenes strain K599 was developed for Meifeng 4,an elite jute variety widely cultivated in China.The transgenic hairy root system for jute was verified by subcellular localization and bimolecular fluorescence complementation(BiFC)assays.The CHLOROPLASTOS ALTERADOS 1(CcCLA1)gene,which is involved in the development of chloroplasts,was targeted for editing at two sites in Meifeng 4.Based on this hairy root transformation,the gRNA scaffold was placed under the control of cotton ubiquitin GhU6.7 and-GhU6.9 promoters,respectively,to assess the efficiency of gene editing.Results indicated the 50.0%(GhU6.7)and 38.5%(GhU6.9)editing events in the target 2 alleles(gRNA2),but no mutation was detected in the target 1 allele(gRNA1)in transgenic-positive hairy roots.CcCLA1 gene editing at gRNA2 under the control of GhU6.7 in Meifeng 4 was also carried out by Agrobacterium tumefaciens-mediated transformation.Two CcCLA1 mutants were albinic,with a gene editing efficiency of 5.3%.These findings confirm that the CRISPR/Cas9 system,incorporating promoter GhU6.7,can be used as a gene editing tool for jute.

Development of an Agrobacterium-mediated CRISPR/Cas9 system in pea(Pisum sativum L.)

Pea(Pisum sativum L.)is an annual cool-season legume crop.Owing to its role in sustainable agriculture as both a rotation and a cash crop,its global market is expanding and increased production is urgently needed.For both technical and regulatory reasons,neither conventional nor transgenic breeding techniques can keep pace with the demand for increased production.In answer to this challenge,CRISPR/Cas9 genome editing technology has been gaining traction in plant biology and crop breeding in recent years.However,there are currently no reports of the successful application of the CRISPR/Cas9 genome editing technology in pea.We developed a transient transformation system of hairy roots,mediated by Agrobacterium rhizogenes strain K599,to validate the efficiency of a CRISPR/Cas9 system.Further optimization resulted in an efficient vector,PsU6.3-tRNA-PsPDS3-en35S-PsCas9.We used this optimized CRISPR/Cas9 system to edit the pea phytoene desaturase(PsPDS)gene,causing albinism,by Agrobacterium-mediated genetic transformation.This is the first report of successful generation of gene-edited pea plants by this route.

Agrobacterium-mediated Transformation of Kentucky Bluegrass

Embryogenic calli of Kentucky bluegrass, named Md, were induced from mature seeds and embryos, and proliferated on medium K3 containing 2,4-dichlorophenoxyacetic acid (2,4-D, 10.0 mumol/L), 6-benzylaminopurine (BAR, 0.5 mumol/L) and K5 which was the K3 medium supplemented with cupric sulfa (0.5 mumol/L) under dim-light condition (20-30 mumol.m(-2).s-1, 16 h light) at 24 degreesC. Embryogenic calli were transformed with plasmids pDM805 Carring bar and gus genes, Which was mediated by an Agrobacterium strain AGL1, four transgenic lines were obtained. The important factors that affect the transformation efficiency and obtain desirable number of transgenic plants included: (1) the quality of embryogenic calli; (2) light condition and time of co-cultivation; (3) concentration of antibiotics used for suppressing the overgrowth of Agrobacterium in the course of transformed plant regeneration; (4) selection pressure, etc. The micro nutrient of cupric had significant influence on the quality of embryogenic calli. This presentation is the first successful protocol of Kentucky bluegrass transformation mediated by Agrobacterium.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

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.

Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon (Citrus Limon (L.) Burm. F. cv. Eureka)

The Arabidopsis ICEI （inducer of CBF expression 1） gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon （Citrus Limon （L.） Burm. F. cv. Eureka） genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome.

In planta Agrobacterium-Mediated Transformation of Rice

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog(MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD_(600) = 0.8–1.0 and the co-cultivation at 25 ℃ for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gus A gene by PCR using the gus A specific primers in the T_0 lines, there were 4 transgenic lines from 286 T_0 lines(1.4% transformation efficiency). However, histochemical glucuronidase(GUS) assay demonstrated that only three of four transgenic lines exhibited gus A expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

Effect of Phytosulfokine-α on Agrobacterium-Mediated Transformation in Rice

Phytosulfokine- α （PSK- α ）, a biologically active peptide acting as a growth factor, plays a key role in cellular differentiation and proliferation. To test if PSK- α has some influence on agrobacterium-mediated transformation in rice, PSK-α at a series of concentrations was added into co-culture medium respectively. The results showed that PSK- α indeed affected the recovery of resistant calli and the transformation frequency of rice varieties Taipei 309 and Lijiangxintuanheigu, PSK- α at the concentration of 10 nmol/L could increase induction of resistant callus and efficiency of transformation, with a 11% and 4.9% top increase, respectively than the control. However, PSK- αat 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effect of PSK-α on agrobacterium-mediated transformation is related with the concentration of 2, 4-D in selection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSK- α plus 2 mg/L 2, 4-D.

Agrobacterium-Mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene

Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cul-tivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with Agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg I/1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis.

Efficient Regeneration and Agrobacterium-mediated Transformation of Brassica napus Cultivar Qingza No.5

Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB ＋ 5 mg/L thidiazuron （TDZ） ＋7.5 mg/L AgNO3 ＋ 0.1 mg/L NAA ＋ 2 mg/L proline （L-pro） ＋ 250 mg/L casein acid hydrolysate （CH） ＋ 3% sucrose; the appropriate growth medium was 1/2 MSB ＋ 1 mg/L IBA ＋ 2 mg/L L-pro ＋ 250 mg/L CH ＋ 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB ＋ 0.2 mg/L IAA ＋ 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.

γ-Al_2O_3高温相变的XRD和Raman光谱比较研究

采用XRD和Raman光谱研究了γ-Al2O3的高温相变。Raman结果表明,位于1 175,1 241 cm-1和1 370,1 400 cm-1的2组谱峰是杂质Fe3+和(或)Cr3+在θ-Al2O3和α-Al2O3环境中产生的荧光光谱,用这2组峰来指认θ-Al2O3和α-Al2O3物相比XRD更灵敏。因此,Raman技术能高灵敏地表征γ-Al2O3的高温相变过程。结果表明γ-Al2O3从800℃开始发生相变,γ-Al2O3同时向θ-Al2O3和α-Al2O3转变,随着温度的升高最终完全转变成α-Al2O3。相变温度与样品本身有关。

Study on the Integration of T-DNA into Lilium longiflorum

[ Objective] The aim of this study is to obtain transgenic Lilium longiflorum Thumb. [ Method] A two-step method of explant and the T-DNA integration technique were employed to transform Lilium longiflorum via Agrobacterium mediated method. [ Result] The best infection effect appeared under the OD600 value of Agrobacterium within 0.6 -0.8, the addition of 250 mg/L AS could increase the transformation efficiency. The optimal concentration of G418 for screening is 50 mg/L. Some putative transgenic plants of Lilium longiflorum with resistance to G418 showed positive in PCR, preliminarily proving that T-DNA gene had integrated into the genome of lily. [ Conclusion] The study may lay a foundation for breeding excellent lily varieties through TDNA integration technique.

教师专业知识转化新探

教师作为"知识工作者",其专业发展水平与知识转化相关联。在理性审视教师知识研究的基础上,立足教师个体自我发展规律,以统整、融通、动态的视角,澄清对学科知识、课程知识、教学知识的新理解,构建了教师专业知识转化的模型。在该模型中,作为知识转化者,教师推动自身知识经历课程化、教学化、学科化三个循环递进过程,进而实现专业知识转化与自我重建。

彩色图像DWT变换下的块SVD零水印

为了提高水印技术的鲁棒性,提出了彩色图像离散小波变换(DWT)下的块奇异值分解(SVD)的零水印.首先对原始载体图像进行离散小波变换,然后选择低频子带进行分块,且对每一块进行奇异值分解,水印则由分解得到的最大前m个奇异值产生.实验结果表明,算法对各种攻击有较强的鲁棒性.

转cry1Ab基因抗虫水稻的培育

采用农杆菌介导的遗传转化法,将cry1Ab基因导入优良三系恢复系明恢63,共获得了92个独立转化株,其中13株为单拷贝。通过发芽试验,获得了11个单拷贝转基因纯合系。ELISA检测结果表明:不同株系其Bt蛋白含量不一样,但杂种的Bt蛋白含量与其亲本一致。室内人工接虫试验及田间抗虫性试验初步表明:5个纯合株系表现高度抗性,且农艺性状与原品种没有显著差异。由此说明,所获得的这些单拷贝转基因株系可作为育种材料,用于抗虫水稻的培育。

基于小波变换和四叉树的图像分形编码算法研究

提出利用小波变换和分形编码的优势,进行图像压缩编码.最低分辨率子带进行失真较小的均匀量化编码.对高分辨率子带设定系数阈值,小波系数小于等于系数阈值则直接置零;大于系数阈值的块采用四叉树算法进行分形编码,如果误差小于等于误差阈值,则记录分形编码参数,如果误差大于误差阈值,则进行四叉树分裂.对算法进行了matlab仿真,实验表明解码图像质量和运算速度都有较大改善.

大学出版社数字出版转型面临的问题及对策分析

在数字化阅读时代,大学出版社数字出版转型势在必行。文章认为由于受重视程度不够、资源整合不足、营利模式不明、社会认同度低、专业人才缺乏等问题的制约,大学出版社数字出版转型仍处于初级阶段和被动的状态中。为更好地适应高等教育数字化战略的要求,大学出版社应因势而动、顺势而为,从提高思想认识、巩固内容优势、创新商业模式、推动融合发展、加强队伍建设等方面入手,积极主动推进数字出版转型。

农杆菌介导bar基因转化水稻胚性愈伤组织的研究

为建立以草丁膦为选择标记的农杆菌Agrobacterium tumefaciens介导的水稻Oryza sativa胚性愈伤组织转化体系,通过农杆菌介导法将bar基因导入了‘日本晴’Oryza sativa‘Nipponbare’‘秀水134’‘Xiushui 134’和‘中花11’‘Zhonghua 11’等3个水稻品种的胚性愈伤组织。在分别以选择压15.0,10.0和10.0 mg·L^(-1)的草丁膦质量浓度进行了3次选择后,聚合酶链式反应(polymerase chain reaction,PCR)发现‘日本晴’‘秀水134’和‘中花11’的愈伤组织转化效率分别是77.9%,67.5%和92.2%,获得的抗性愈伤组织可以进一步分化成苗。它们的分化率依次是76.1%,44.4%和86.7%;成苗率分别为65.6%,66.7%和30.7%。在以草丁膦作为筛选标记时,首选‘日本晴’作为转化受体进行遗传转化。该体系为以抗除草剂基因作为筛选标记的水稻遗传转化和基因功能验证提供了技术基础,获得的抗草丁膦转化植株为水稻育种提供了材料。

试论当代生态人类学理论的转向

本文在回顾人类学生态研究的基础上,论述了近20多年来西方生态人类学理论发展的新趋势———环境人类学,并提供了环境人类学研究的具体案例。认为环境人类学所具有的综合性、批判性和参与性等特点,使它区别于传统生态人类学。

GPS-RTK界址点测量系统性误差改正方法探讨

在GPS-RTK测量界址点时,通过点校正获取坐标转换参数会引起系统性的坐标转换残差;在测定建筑物墙角等界址点时,只能以接收机天线的外缘靠近墙角位置,使得天线中心偏离界址点的实际位置,导致界址点测量偏心差。为此,通过在测区所有已知点上检测其坐标以建立测区的坐标转换残差改正模型,并导出三种基于天线偏心改正的界址点坐标计算及其误差公式,基本消除RTK界址点测量中的系统性误差影响。实际应用表明,该方法原理简单且便于外业施测和编程实现,可提高GPS-RTK界址点测量的精度。