植物营养的分子生物学和遗传学研究方法课件.ppt

1、植物营养的遗传学和分子生物学植物营养的遗传学和分子生物学研究方法研究方法高亚军高亚军2009-04-09植物营养学研究方法（植物营养学研究方法（2）养分资源的高效利用养分资源的高效利用光照、温度、光照、温度、降水、降水、COCO2 2浓度浓度等气候因素等气候因素土壤土壤因素因素？环境条件环境条件的影响的影响施肥施肥改善改善土壤条件土壤条件合理合理施肥施肥植物体本植物体本身的特性身的特性高效高效肥料肥料冬小麦不同品种氮素利用效率的差异冬小麦不同品种氮素利用效率的差异0102030405060708090西杂1 号西农1 3 76（父）陕1 50小偃6 号西农2 6 11西农1 3 76（母）陕1

2、 60阎麦8 9 11矮丰3 号陕2 29氮素利用率（%）油菜不同基因型氮素吸收效率油菜不同基因型氮素吸收效率具有显著差异具有显著差异 你见过蓝色的玫瑰、你见过蓝色的玫瑰、百合、康乃馨、菊花、百合、康乃馨、菊花、郁金香和蓝色的月季花郁金香和蓝色的月季花吗？吗？你见过蓝色的玫瑰、百合、康乃馨、你见过蓝色的玫瑰、百合、康乃馨、菊花、郁金香和蓝色的月季花吗？菊花、郁金香和蓝色的月季花吗？玫瑰、百合、康乃馨、菊花、郁金香和月季等玫瑰、百合、康乃馨、菊花、郁金香和月季等花卉没有天然的蓝花品种。花卉没有天然的蓝花品种。因为它们缺乏因为它们缺乏黄酮黄酮-35-羟化酶羟化酶，因而不能产，因而不能产生生翠雀素翠

3、雀素。翠雀素翠雀素是是花青素花青素的一种，呈紫色。的一种，呈紫色。花青花青素素是花卉中控制花色的主要化学成分之一。是花卉中控制花色的主要化学成分之一。Plant Genetic Modification植物遗传修饰植物遗传修饰基本概念基本概念转基因植物（transgenic plant）转化植物（transformed plant）基因工程植物（genetically engineered plant）遗传修饰植物（genetically modified plant）Why use Genetic Modification?为什么进行遗传修饰？为什么进行遗传修饰？uAccelerate th

4、e breeding process加速育种进程加速育种进程 Introduce/enhance desired trait in an established genetic backgrounduExtend the gene pool扩大基因库扩大基因库 Select genes from any Kingdom What are the Uses of GM Plants?遗传修饰植物做什么用？遗传修饰植物做什么用？uResearch Largest number of transgenic plants are currently created for research purpo

5、ses Knock-outs,over-expression,modified proteinsK.Yamaguchi-Shinozaki,JIRCAS,Japan stress-induciblepromoter drivingdrought-andcold-responsivetranscription factorwild typeWhat are uses of GM Plants?uCommercial Applications 商业用途商业用途 Altered agronomic traits改变农学性状改变农学性状 Disease/insect resistance Virus

6、resistance Herbicide resistance Salt/drought tolerance Cold tolerance Enhanced yields,other quantitative traits PhytoremediationApplication of Roundup herbicideField following application time2019Nature Biotechnology 25:271(2019)Nature Biotechnology 25:271(2019)Nature Biotechnology 25:271(2019)Other

7、 uses of GM Plants?uFunctional foods(humans and livestock)Today:Golden rice Vitamin A enriched Future directions:Boosted antioxidants Elevated content of specific minerals Removal of food allergens,carcinogensGreater public acceptance when the technology is shown to more greatlybenefit consumers?转基因

8、高铁水稻转基因高铁水稻由日本、韩国和丹麦研究人员组成的研究小组由日本、韩国和丹麦研究人员组成的研究小组13日宣日宣布，他们成功培育出铁含量布，他们成功培育出铁含量3倍于普通稻米的水稻，倍于普通稻米的水稻，并证实这种高铁水稻能改善实验鼠的贫血症状。并证实这种高铁水稻能改善实验鼠的贫血症状。从水稻中普遍含有的全草含烟胺（从水稻中普遍含有的全草含烟胺（nicotianamine）着手，对水稻品种进行改良。全草含烟胺是一种氨基着手，对水稻品种进行改良。全草含烟胺是一种氨基酸，它能与铁元素相结合，将土壤中的铁元素从水稻酸，它能与铁元素相结合，将土壤中的铁元素从水稻的根部输送到稻穗。研究人员通过转基因技术

9、，把能的根部输送到稻穗。研究人员通过转基因技术，把能够提高全草含烟胺合成能力的基因植入水稻内，令水够提高全草含烟胺合成能力的基因植入水稻内，令水稻合成全草含烟胺的量增加到普通水稻的稻合成全草含烟胺的量增加到普通水稻的9.6倍，提高倍，提高了水稻对铁的吸收能力和运输能力，培育出了铁含量了水稻对铁的吸收能力和运输能力，培育出了铁含量3倍于普通稻米的水稻。倍于普通稻米的水稻。转基因高铁水稻转基因高铁水稻研究人员用来自高铁水稻的糙米喂养患有研究人员用来自高铁水稻的糙米喂养患有贫血症的实验鼠，两周后，实验鼠血液内贫血症的实验鼠，两周后，实验鼠血液内的血红蛋白浓度恢复到正常水平。的血红蛋白浓度恢复到正常水

10、平。研究人员表示，所有植物中都有全草含烟研究人员表示，所有植物中都有全草含烟胺，因此，相同技术也应该能够用于培育胺，因此，相同技术也应该能够用于培育铁含量丰富的其他作物。铁含量丰富的其他作物。铁是生物必需的营养，据世界卫生组织的铁是生物必需的营养，据世界卫生组织的报告，全球约一半人口缺铁。报告，全球约一半人口缺铁。How to Modify a Plant（如何修饰植物？）Initial Stages（准备工作）1.Final goal（确定目标）2.Trait of interest（感兴趣的性状）3.Understanding the biology（了解生物学背景）4.Source of

11、 trait/gene of interest（性状来源/感兴趣的基因）5.Site of gene expression（基因表达位点）6.Vector（载体）7.Selection of transformation method（转化方法选择）性状u质量遗传性状（qualitative genetic trait）：由单基因控制的性状，如抗虫、抗病、抗除草剂等生物胁迫性状及耐寒、耐旱和耐暑等非生物胁迫性状。u数量遗传性状（quantitative genetic trait）：由多基因控制的性状，如优质高产性状。Plant ModificationGetting to work（工作步骤

12、）1.Cloning gene（克隆目的基因）2.Plant transformation（植物转化）3.Selection of transgenic plants（转基因植物的筛选）4.Screening for appropriate expression levels（适宜表达水平的筛选）5.Isolation of homozygous lines（纯合体的分离）6.Testing of plants（植物鉴定）Plant ModificationGene of interest（感兴趣的基因）Endogenous gene is your best choice Codon usa

13、ge is important for non-plant genes Selection of cDNA versus genomic clones(n/a for RNAi)Plant ModificationCloning gene（克隆目的基因）Source borrow,buy or build your own Involves amplification of the gene using PCR and insertion into an intermediate vector(often)Sequence verification Modifications can be m

14、ade at this point Site directed mutagenesisPlant ModificationTransfer of gene into the binary vector（将目的基因导入双元载体）Binary vectors often difficult to work with Low copy number Large size 10 kb Generally lack LacZ based screening(blue/white)Verification of insert can be checked by colony PCR and sequenc

15、ingPlant Modification Transformation method（转化方法）1.Agrobacterium-mediated transformation（农杆菌介导的转化）is the most common technique(Electroporation directly into Agro strain)2.Electroporation（电穿孔法/电激法）3.Biolistic transformation “Gene gun”（基因枪）Plant ModificationCo-cultivation and primary selection（转基因植物的筛

16、选）Co-cultivate leaf discs/fragments with Agrobacterium culture Wash away excess culture Incubate in shoot regeneration medium w/antibiotics-1(bacterial elimination)Incubate in shoot regeneration medium w/antibiotics-2(selection for transgenic plant cells)Plant ModificationObtaining homozygous lines（

17、纯合体的获得）All T1 plants will be heterozygous(or more correctly,“hemizygous”).Some T2 plants will be homozygous Because we do not know the site of integration we must look at germination rates of T3 seed to determine zygosity of T2 parents Hemizygous 3:1 progeny on selective media Homozygous All progeny

18、 germinate on selective media Plant ModificationTesting of plants（植物鉴定）Depends on desired goal Screen:Expression level Protein production level Presence of trait Enhancement of trait Fitness level of plants寻找决定某重要性状的基因基因功能分析Why sequence genomes?为什么进行基因组测序？为什么进行基因组测序？The genome sequence provides:accu

19、rate genome sizenumber and type of genesgene/genome structuretools for investigating gene function.Genome Sequence of Multicellular Organisms First completed for:Caenorhabditis elegans2019Drosophila melanogaster2000Arabidopsis thaliana2000Why were these organisms chosen?Small sizeLarge number of pro

20、genyShort generation timeSmall genome(50-150 Mbp)Scope of the problem All genes in Arabidopsis have been identified through genome sequencing so cloned genes are readily accessible.But of 27,000 genes:#genesKnown biochemical/cellular/biological function 1,000Predicted biochemical function from annot

21、ation16,000Unknown10,000Using Mutants to study Biological processes1.What is a mutant（突变体）（突变体）?Mutant:an individual,organism,or new genetic character arising or resulting from an instance of mutation,which is a sudden structural change within the DNA of a gene or chromosome of an organism resulting

22、 in the creation of a new character or trait not found in the wild-type.The natural occurrence of genetic mutations is integral to the process of evolution.Wild-type:the genetic term used in texts for the typical form of an organism,strain,gene,or characteristic as it occurs in nature.WikipediaMutan

23、ts2.Where do mutants come from?Natural occurrenceMan-made3.How to make mutants?Radom mutagenesis:l Chemical(point mutations):EMS（甲基璜酸乙酯）、（甲基璜酸乙酯）、nitrosoguanidine（亚硝基胍亚硝基胍）l Irradiation(deletions):Gamma radiation,UV,Xray,fast neutronl DNA insertions(insertional knockouts):transposons（转座子：一种脱氧核糖核酸片断，

24、它能够移到同一个或另一个转座子：一种脱氧核糖核酸片断，它能够移到同一个或另一个染色体、质体或细胞上的新位置，并转录各种基因的特性，如对抗菌体的抵染色体、质体或细胞上的新位置，并转录各种基因的特性，如对抗菌体的抵抗力抗力）,T-DNADirectional mutagenesis:1)Site-directed mutagenesis（定点诱变）（定点诱变）2)Gene silencing:RNA interference(RNAi),micro-RNA 4.What is a loss-of-function mutation?Loss-of-function mutation:gene pr

25、oduct has less or no function.Phenotypes associated with such mutation are most often recessive.5.What is a gain-of-function mutation?Gain-of-function mutation:change the gene product such that it gains a new and abnormal function.These mutations usually have dominant phenotypes.6.What is forward ge

26、netics（正向遗传）（正向遗传）?Forward genetics:a genetic screen for new genes.phenotype（表型）（表型）genotype（基因型）（基因型）Mutants7.What is reverse genetics（反向遗传）（反向遗传）?Reverse genetics:an approach to discovering the function of a gene.Gene phenotype 1 attttttttg tttctctcaa taatatttgt gttcatactg tttcgctgtt tccaatacat 61

27、 actaatggat aacacaaacc gtcttcgccg ccttcactgt cataaacaac ccaagttcac 121 tcatagctct caagaagtga gtagtatgaa atgggagttt atcaatatga ccgaacaaga 181 agaagatctc atctttagaa tgtacagact tgttggcgac aggtgggatt taatagcaag 241 aagagtggtg ggacgtgagg caaaggagat agagagatac tggattatga gaaattgtga 301 ctatttctcc cacaaatg

28、ac gcaaaagtca ctctctttaa aaaatctcga gaatctccct 361 gtttttctat ttctcatcct cattaaataa tttctgtgat tgtggacaag aaaagcttta 421 ttaagttgtc gagtggggtt ctcct Reverse genetics1:Structural analysis of the gene 2:Phenotypic analysis of the mutant3:Analysis of gene expression4:Phenotypic analysis of the gene overexpression（过量表达）（过量表达）transgenic lines