The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.

A Theory of Bio-Quantum Genetics

The physical mechanism of heredity or inheritance of genes is a quantum mechanical and/or quantum computational process. A theory of bio-quantum genetics is established in this paper. Principle of Bio-quantum Genetics is suggested. I propose and define the soft-genes of genetics controlling the processes of heredity or inheritance of genes. This research deals with the quantum mechanisms of Mendel plant heredity and family inheritance as examples of bio-quantum genetics, deepening our understanding of heredity or inheritance. I believe that more contributions will be made to promote researches of bio-quantum genetics or quantum biology at large.

人类“绿色基因”假说(Human"Green-Gene"Hypothesis):核心内容、科学佐证与实践意义

首先,对人类“绿色基因”的形成进行探讨,包括人类(动物)与植物共同维持大气中碳氧平衡、人类(动物)与植物互为食物链关系、人体功能中存在植物性功能、绿色是人类眼睛最易看到的颜色、人类血红蛋白的结构相似于叶绿素结构、作为人体呼吸器官的肺部相似于树木地上部形状(树形)、人类肠道绒毛相似于植物根系毛细根等方面。进而对支持早期人类生活的植物类型与栽培植物诞生进行研究。在此基础上,提出人类“绿色基因”假说及其核心内容,归纳该假说的科学佐证。人类“绿色基因”假说从整体的、发展的思维解释人类与植物关系,奠定了园艺疗法、园林康养、森林康养的坚实基础,并指出接触植物、进行园艺操作与田园劳作是人们实现接地、激发触觉潜在功能的途径。

Response Mechanism of Plants to Drought Stress

Drought stress is an important factor affecting plant growth and development.It will provide a theoretical basis for cultivating new stress-resistant varieties and improving water utilization rate of plants by studying the regulation mechanism of osmotic adjustment and water transportation under drought stress,and understanding the physiological and biochemical characteristics and stress resistance mechanism.

Consensus maps of cloned plant cuticle genes

Plant cuticle,which covers the plant surface,consists of waxes and cutins,and is associated with plant drought,cold,and salt resistance.Hitherto,at least 47 genes participating in the formation of plant cuticle have been cloned from Arabidopsis thaliana,Oryza sativa,Zea mays,Ricinus communis,Brassica napus,and Medicago truncatula;and about 85% of them encode proteins sharing above 50% identities with their rice homologous sequences.These cloned cuticle genes were mapped in silico on different chromosomes of rice and Arabidopsis,respectively.The mapping results revealed that plant cuticle genes were not evenly distributed in both genomes.About 40% of the mapped cuticle genes were located on chromosome 1 in Arabidopsis,while 20% of the mapped cuticle genes were located on chromosome 2 but none on chromosome 12 in rice.Some cloned plant cuticle genes have several rice homologous sequences,which might be produced by chromosomal segment duplication.The consensus map of cloned plant cuticle genes will provide important clues for the selection of candidate genes in a positional cloning of an unknown cuticle gene in plants.

Transient Expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine （PEI） nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5：1-1：4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P〈5 and reached the highest at N/P=5, and began to decrease beyond N/P〉5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.

Construction of Plant Expresion Vector Carrying Two Insecticidal Genes and Obtain Insect-resistant Transgenic Tobacco Plants

１．ＩｎｔｒｏｄｕｃｔｉｏｎＣｒｏｐｐｌａｎｔｓａｒｅｓｕｓｃｅｐｔｉｂｌｅｔｏａｔａｃｋｂｙａｗｉｄｅｒａｎｇｅｏｆｈｅｒｂｉｖｏｒｏｕｓｉｎｓｅｃｔｓ．Ｉｔｉｓｅｓｔｉｍａｔｅｄｔｈａｔａｒｏｕｎｄ１３％ｏｆｔｈｅｔｏｔａｌｗｏｒｌｄｗｉｄｅ...

Cloning of XET gene from Anthocephalus chinensis and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene （XET）, abundantly expressed in the cambium of Anthocephalus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame （ORF） encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment ofAcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism ofAcXET gene during wood formation.

Genome-wide Identification and Analysis of MVD Gene Family in Euphorbiaceae Plants

The mevalonate diphosphate deearboxylase （MVD） is an essential enzyme in mevalonate （MVA） pathway that catalyzes the irreversible Mg2＋ -ATP de- pendent decarboxylation of 6-carben compound mevalonate-5-diphosphate （MVAPP） into 5-carbon isopentenyl diphosphate （ IPP）, the building block of sterol and isoprenoid biosynthesis. In this study, based on the published geanme sequences and ESTs, a genome-wide search was carried out for the first time to identify MVD gene family in four genome-sequenced Euphorbiaceae plants, i.e. castor bean （ Ricinus communis）, physic nut （ Jatropha curcas）, cassava （Manihot esculenta） and rubber tree （Hevea brasiliensis）, and to analyze the gene structure and phylogenetic characteristics. According to the experimental results, 1, 1,2 and 2 MVD genes, which all contain 9 introns, were identh＇ied from castor bean, physic nut, cassava and rubber tree, respectively. Homology analysis indicates that MVD genes are widely distributed in eukaryotes, some archaea and eubacteria, which suggests an early origin of this gerte family. Although MVD genes were identified in most green plants, no homologous genes were found in unicellular green algae. In most genome-sequenced plants including castor bean and physic nut, a single copy of MVD gene was found; however, in cassava and rubber tree, two copies were identified just like that in moss, maize, Arabidopsis and poplar. ＂In castor bean, digital expression profiling suggests that in five examined tissues, i.e. leaf, flower, II/III stage endosperm, V/VI stage endosperm and seed, RcPMK was expressed strongly in flower and II/III stage endosperm, moderately in V/VI stage endosperm and leaf, and weakly in seed.

Seedling and adult plant resistance to leaf rust in 46 Chinese bread wheat landraces and 39 wheat lines with known Lr genes

Wheat leaf rust,caused by Puccinia triticina(Pt),is an important foliar disease that has an important influence on wheat yield.The most economic,safe and effective way to control the disease is growing resistant cultivars.In the present study,a total of 46 wheat landraces and 34 wheat lines with known Lr(leaf rust resistance)genes were inoculated with 16Pt pathotypes for postulating seedling resistance gene(s)in the greenhouse.These cultivars and five wheat differential lines with adult plant resistance(APR)genes(Lr12,Lr22b,Lr34,Lr35 and Lr37)were also evaluated for identification of slow rusting resistance in the field trials in Baoding,Hebei Province of China in the 2014–2015 and 2015–2016 cropping seasons.Furthermore,10 functional molecular markers closely linked to 10 known Lr genes were used to detect all the wheat genotypes.Results showed that most of the landraces were susceptible to most of the Pt pathotypes at seedling stage.Nonetheless,Lr1 was detected only in Hongtangliangmai.The field experimental test of the two environments showed that 38 landraces showed slow rusting resistance.Seven cultivars possessed Lr34 but none of the landraces contained Lr37 and Lr46.Lr genes namely,Lr9,Lr19,Lr24,Lr28,Lr29,Lr47,Lr51 and Lr53 were effective at the whole plant stage.Lr18,Lr36 and Lr45 had lost resistance to part of pathotypes at the seedling stage but showed high resistance at the adult plant stage.Lr34 as a slowing rusting gene showed good resistance in the field.Four race-specific APR genes Lr12,Lr13,Lr35 and Lr37 conferred good resistance in the field experiments.Seven race-specific genes,Lr2b,Lr2c,Lr11,Lr16,Lr26,Lr33 and LrB had lost resistance.The 38 landraces showed slow rusting resistance to wheat leaf rust can be used as resistance resources for wheat resistance breeding in China.

Cloning the Promoter of BcNA1 from Brassica napus and Fad2 Gene from Arabidopsis thaliana and Construction of the Plant Expression Vector

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Plant Long ncRNAs: A New Frontier for Gene Regulatory Control

Long non-coding RNA (lncRNA) refers to an over 200 nt functional RNA molecule that will not be translated into protein. Previously thought to be dark matters of the genome, lncRNAs have been gradually recognized as crucial gene regulators. Although tremendous progress has been made in animals and human, the study of lncRNAs in plant is still in its infancy. Here, we reviewed the biogenesis and regulation mechanisms of lncRNAs and summarized the achievements that have been made in plant lncRNA identification and functional characterization. Genome-wide identification has uncovered large amount of lncRNAs in Arabidopsis, Rice, Maize and Wheat, and more information from other plant species will be expected with the aid of deep sequencing technologies. Similar to other species, LncRNA-mediated gene regulation also widely exists in plants, even though only a few functionally characterized examples are available. Up to now, at least four divergent lncRNA-mediated regulation mechanisms have been unraveled, including target mimicry, transcription interference, PRC2 associated histone methylation and DNA methylation. lncRNAs may be involved in the regulation of flowering, male sterility, nutrition metabolism, biotic and abiotic stress response in plants.

Leafy head formation of the progenies of transgenic plants of Chinese cabbage with exogenous auxin genes

The experiment was performed to evaluate the progenies of plant lines transgenic for auxin synthesis genes derived from Ri T-DNA. Four lines of the transgenic plants were selfcrossed and the foreign auxin genes in plants of T5 generation were confirmed by Southern hybridization. Two lines, D1232 and D1653, showed earlier folding of expanding leaves than untransformed line and therefore had early initiation of leaf y head. Leaf cuttings derived from plant of transgenic line D1653 produced more adventitious roots than the control whereas the cuttings from folding leaves had much more roots than rosette leaves at folding stage, and the cuttings from head leaves had more roots than rosette leaves at heading stage. It is demonstrated that early folding of transgenic leaf may be caused by the relatively higher concentration of auxin. These plant lines with auxin transgenes can be used for the study of hormonal regulation in differentiation and development of plant organs nd for the breeding of new varietywith rapid growth trait.

Rabbit Defensin(NP-1) Gene Transformation of Wheat and In Vitro Microbicidal Activity Assay of Transgenic Plants

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Obtaining-High Pest-resistant Tobacco Plants Carrying B.t. insecticidal Gene

0 ＩｎｔｒｏｄｕｃｔｉｏｎＢｒｅｅｄｉｎｇｐｅｓｔｒｅｓｉｓｔａｎｔｐｌａｎｔｓｕｓｉｎｇｐｌａｎｔｇｅｎｅｔｉｃｅｎｇｉｎｅｅｒｉｎｇｔｅｃｈｎｉｑｕｅｉｓａｎｅｆｆｅｃｔｉｖｅｓｔｒａｔｅｇｙｉｎｔｈｅｉｎｔｅｇｒａｔｅｄｐｅｓｔｍａｎａｇｅｍ?..

The design of aided software for osmotic stress responding genes mining in plant genome

A software and algorithm which based on random sequence model uses osmotic stress responding cis elements from existing information sources of biology was designed. It can infer the genic downstream function of Arabidopsis thaliana through analyzing its promoter region, and can offer effective aided analysis to mine osmotic stress responding genes in Arabidopsis thatiana genome. The practical application proves that this software can aid to analyze vast genic data and offer important data evidence.

Changes in Gene Expression in Needles and Stems of Pinus radiata Rootstock Plants of Different Ontogenic Age

A major problem in forest clonal productivity is the loss of morphogenetic capability with the increasing age of plants. However, despite of the importance of loss of morphogenetic competence, very little research has been done about the underlying mechanisms involved in this process. For this reason, a gene expression analysis using dot blot technique was performed in needles and stems of 1- and 3-year old Pinus radiata rootstock plants with a proved decrease in morphogenetic competence. Needles of one year old rootstock plants showed a higher number of up-regulated in genes mainly corresponding to photosynthesis and protein synthesis, degradation and modification, reflecting a higher number of active pathways in younger hedges, contrary to the older ones. Gene expression profiles found in stems are in agreement with those found in needles, indicating more active pathways in younger rootstock plants than in older ones. Several transcripts regulating transcription and translation were up-regulated in young competent tissues. Three-year-old stems presented an increase in the expression of an ethylene response factor, involved in plant organ senescence, indicating that pathways involved in senescence and ageing might inhibit the adventitious root formation, as in the older cuttings.

Enhanced Stem Nematode Resistance of Transgenic Sweetpotato Plants Expressing Oryzacystatin-I Gene

Enhanced stem nematode resistance of transgenic sweetpotato （cv. Lizixiang） was achieved using Oryzacystatin-I （OCI） gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.

Horizontal gene transfer of plant-specific leucine-rich repeats between plants and bacteria

Leucine rich repeats (LRRs) are present in over 14,000 proteins that have been identified in viruses, bacteria, archaea, and eukaryotes. Two to sixty-two LRRs occur in tandem forming an overall arc shaped domain. There are eight classes of LRRs. Plant specific LRRs (class: PS-LRR) had previously been recognized in only plant proteins. However, we find that PS-LRRs are also present in proteins from bacteria. We investigated the origin of bacterial PS-LRR domains. PSLRR proteins are widely distributed in most plants;they are found in only a few bacterial species. There are no PS-LRR proteins from archaea. Bacterial PS-LRRs in twenty proteins from eleven bacterial species (in the three phyla: Proteobacteria, Cyanobacteria, and Bacteroidetes) are significantly more similar to the PS-LRR class than to the other seven classes of LRR proteins. Not only amino acid sequences but also nucleotide sequences of the bacterial PS-LRR domains show highly significant similarity with those of many plant proteins. The program, EGID (Ensemble algorithm for Genomic Island Detection), predicts that Synechococcus sp. CYA_ 1022 came from another organism. Four bacterial PS-LRR proteins contain AhpC-TSA, IgA peptidase M64, the immunoglobulin domain, the Calx-b domain, and the He_PIG domain;these domains show no similarity with any eukaryotic (plant) proteins, in contrast to the similarities of their respective PS-LRRs. The present results indicate that horizontal gene transfer (HGT) of genes/gene fragments encoding PS-LRR domains occurred between bacteria and plants, and HGT among the eleven bacterial species, of the three phyla, as opposed to descent from a common ancestor. There is the possibility of the occurrence of one HGT event from plant to bacteria. A series of HGTs might then have occurred recently and rapidly among these eleven species of bacteria.

Cloning of Promoter of Chinese Bean GRP 1.8 Gene and Characterization of Its Function in Transgenic Tobacco Plants

In order to learn the expression pattern of GRP1 8(glycine rich protein) gene promoter in transgenic plants and to explore its potential application in plant genetic engineering for vascular specific expression of interested genes, GRP 1 8 promoter was amplified by PCR from Chinese bean genomic DNA. The intermediate vector was constructed by inserting vascular specific expression promoter of GRP 1 8 gene in vector pBI 101. The regenerated tobacco plants obtained were analyzed by PCR to select the putative transgenic plants. The histochemical localization of GUS( β D glucosidase) activity indicates that as for that of GRP 1 8 promoter we can confer the vascular specific expression of GUS gene.