1、大丽轮枝菌激活蛋白VdAL(维大利)的创制与功能研究 Development and Function study of activator protein VdAL from Verticillium dahliaJunsheng Qi；Zhizhong GongChina Agricultural University,College of Biological SciencesState Key Laboratory of Plant Physiology and B； Yinzhong Cui；Haitao QianPherobio Technology Co.,L http:/ Co

2、ntents lWhat is VdAL（来源及性质）lHow VdAL was produced（生产方法）lHow to use VdAL and its optimum concentrations（推荐用法）lVdAL induces resistance to disease and abiotic stresses（抗病）lVdAL promotes crop growth and increase yield（增产）lVdAL promotes fruit maturation and improve quality（改善品质）lThe possible mechanism of

3、 VdAL（作用机理）What is VdAL(维大利)?CottonStraw berry Egg plant Sun flowerVdAL:A secreted protein from Verticillium dahlia(大丽轮枝菌),which is a fungal pathogen that causes vascular wilt diseases in many plant species,such as cotton,straw berry,egg plant,sun flower.Qi Junsheng Gong ZhizhongVdAL is an ASPF2-lik

4、e secreted protein with 297 AAYTK12 pSUC2YTK12 pSUC2-MSP-MSP pSUC2-VdAL pSUC2-VdALTTCTTCYPRAAYPRAACMDCMD-W-WYPDA YPDA VdAL:Verticillium dahliae Asp-f2 LikeVdAL is a zinc ion binding protein（锌离子结合蛋白）WT line 2 line 3WT line 2 line 3Line 2 line 3 WTVdALUBQDisease Index(%)020406080100*WT line 2 line 3AC

5、BDExpression of VdAL increases disease resistance to Verticillium in Arabidopsis （拟南芥表现抗黄萎病）VdALLine 2VdALLine 3WTVdAL treatment induce the resistance to Verticillium wilting （诱导棉花对黄萎病抗性）CK72 h144 hThe cotton leaves were first treated with VdAL,and then the petioles were soaked in the Verticillium s

6、olution for different times.Plant vaccine？植物疫苗？treatmentCKtreatment VdAL patents：3 Chinese national invention patents 3项国内专利 共申请共申请13个国家专利保护。澳大利亚、哈萨克斯坦、俄罗斯已授权个国家专利保护。澳大利亚、哈萨克斯坦、俄罗斯已授权VdAL patents：2 PCTs applied in:Australia、Brazil、Canada、EU、India、Russia、South Africa、America、Chile、Indonesia、Kazakhsta

7、n、Sri Lanka、Thailand How was VdAL produced？（生产方法）？（生产方法）E.coli Gene Engineering StrainsProtein powder;Molecular weight 30 KD;1-5%content;Water soluble;Working concentration 1-5 ppm;Quality guarantee period:2 years E.coli liquid fermentation（液体发酵）（液体发酵）VdAL is very stable for heat.VdAL bio-activity t

8、est on wheat seed germination（蛋白活性生测）VdALP-2 1ppmVdALP-1 1ppmddH2OVdAL powder 1ppmThe effect of VdAL pure protein(90%)for wheat seed germination VdAL Naming and toxic testing（毒性检测）（毒性检测）Acute Toxicity Test：以ICR小鼠为研究系统。结果显示该受试物对 ICR种雌、雄小鼠的经口半数致死剂量 LD50 5000 mg/kg BW。该样品属于实际无毒级物质化学命名：大丽轮枝菌激活蛋白LD50 500

9、0 mg/Kg BW,nontoxicVerticillium dahlia activator protein1.Foliage spraying:Apply on leaf surfaces of different leaf vegetables and various fruit vegetables;of various crops such as rice,wheat,cotton,soybean and rape;of fruit trees such as red grape,Chinese date and cherry etc.2.The suggesting sprayi

10、ng time:Spray one time in the seedling stage and one time in the flowering stage with 3-7000 time dilution solution for above plants.For wheat and rice,spraying one time at heading stage with 10000 time dilution solution.3.Seed soaking:5000-8000 time dilution solution for vegetables,cotton and soybe

11、an;10000 time dilution solution for wheat,rice and corn.4.Seed coating:1-2%VdAL mixed uniformly with the seed coating agents will significantly increase seedling emergence rate and strength,enhance the drought tolerance(for example,wheat seeds).How to use VdAL？（推荐用法）VdAL can be used as a mixture wit

12、h different spraying pesticides,fertilizers or other solutions.VdAL induces disease resistance（诱导抗病性，无直接抑菌活性）A:VdAL itself show no effect on pathogens(Rhizopus Niger)；B:Germinating effct after wheat seed soaked in 1-2ppm VdAL for 6-12hAB CK1ppmS2ppmD2ppmPSeed coating with VdAL induces the wheat drou

13、ght resistance(2015-10,Zhuozhou,Hebei）（小麦种子处理抗旱）CK 0.2%+福福 1%+福福 2%+福福 4%+福福CK 1%+福福 2%+福福 6%福戊福戊 CK 0.2%+福 1%+福 2%+福 4%+福 ACBA&B&C：6%seed coating agent adding 1%-2%VdAL protein can significantly improve the drought resistance of wheat.CK1/20000 VDAL seed soaking（2018,JILIN,JIAN）VDALCKVDAL叶尖黄化(高温胁迫)

14、防止播种前根芽过长叶尖不黄化(高温胁迫)浸种三天浸种六天播种后第七天 VdAL induces the rice high-temprature resistance（水稻种子处理抗高温）VdAL induces the vegetable cold resistance（蔬菜叶面喷施抗寒）VdAL seed soaking+spray induce the resisrence of spring frozen（2018-6，Xinxiang Henan）百农4199（小麦抗倒春寒）1%VdAL+seed coating seed coatinging cktreatmentgrains p

15、er ear 1000-grain weight（g）spike number per sq.m.grain weight per sq.m.yield per unit area（mu）CK18.755.2644.920.64464.3355.52398.3337.86265.69seed coatig 24.401.9546.981.94510.3323.59496.6730.55331.281%VdAL seed coating25.281.2752.052.89491.3318.88475.0098.49316.83（+19.24%）（-4.36%）原粉（1g/亩）spray25.75

16、5.4746.220.85486.0040.84420.0056.79280.14（+5.44%）（-15.44%）seed coating+原粉 spray30.432.9147.771.37443.6727.02450.0082.31300.15（+12.97%）（-9.40%）1%VdAL seed coating+原粉spray27.932.1748.453.37523.3326.57563.3376.87375.74（+41.42%）（+13.42%）VdAL delays rice senescence in China（延缓水稻叶片衰老）Liaoning province 201

17、8.10Henan province 2018.10ckckVdALVdALVdAL increases sheath blight(纹枯病)resistance when sprayed at early flowering time dd treatment15 days after 1st spray15 days after 2nd spraydieseaseindexcontrol efffct（%）Significant differencedieseaseindexcontrol efffct（%)Significant difference嘧菌酯0.6788.4ABb3.257

18、8.5ABb吡唑醚0.9484.1Bc4.3971.0BcVdAL0.9683.8Bc4.6469.8BcV+嘧0.4592.3Aa2.3284.8AaCK5.97-15.22-Azoxystrobin Azoxystrobin+VdAL（2016-9，Changsha,Hunan，0.5ha，Indica）（抗纹枯）PyraclostrobinVdALCKVdAL increases sheath blight resistance（抗纹枯）（2017-10，Hangzhou，1.8ha，CNRRI）The occurrence of sheath blight tended to be l

19、ighter with the increase of VdAL concentration(A).Analysis showed that the control effect of VdAL on sheath blight reached a significant level(2 ppm)or very significant(3ppm)(B).0 00404050506060CKP03P05P10Index of Sheath blightCK 1ppm 2ppm 3ppm ABCK 1ppm 2ppm 3ppm 13.548.03平均病级：5级 3级 2级 1

20、级VdAL increases sheath blight resistance（2018，Xinxiang，Henan）黄金晴（抗纹枯）VdAL field resistance evaluation on rice against false smut(抗抗稻曲病稻曲病)0 00404050506060CKCK1/100001/10000Disease paniclesl B&C:The number of diseased Panicles in the control group was 6.15 on average,but only 3.38 in the t

21、reatment group,and the degree of disease prevention was lighter than that in the control groupl A：Field growing conditionsABCck 1/10000VdALVdAL increases rice blast resistance（2018，Fangzheng，Heilongjiang）龙洋16（抗稻瘟病）VdAL increases sweet potato insect resistance（2017，Handan，Hebei）（抗叶甲取食）CK 1/1000 1/300

22、0 1/5000 1/10000 VdAL promotes wheat seed germination(促进小麦发芽)VdAL promotes rice seed germination （2018-6，Beijing，soaking 7 days）（种子促进）TreatmentPlant heightRoot lengthIncreasing rate（root length）（%）CK3.000.32Y200002.361.07 234%Y250002.780.48 50%Z250002.610.81 153%Y300002.700.53 66%VdAL represses rice

23、 seed germination（2018-6，Beijing，soaking 8 days）（种子抑制）ckY 500Z 500Z 333Z 250l The powder 1g and the preparation 0.67g have the same effect,but the germination potential of the preparation 0.67g is more neat than that of the powder 1g.l 1g and 1.34g preparations have high germination rate,better inhi

24、bition effect;when the use of 1g preparations has reached a certain degree of inhibition,the increase in dosage will not increase the inhibition effect.VdAL seed treatment promotes seed germination and seedling emergence（促进种子萌发）corn rice wheat soybean VdAL seed treatment for strong seeding（2018，Yans

25、hou，Heilongjiang）北稻4（浸种壮苗）VdAL foliage spraying increases yield of peanut（2018，Shenyang，Liaoning）（叶面喷施花生增产）VdALCKTreatmentCK1/3300 VDALBoll Number Per Plant5.65.9Single boll weight（g）5.425.5Lint percentage（%）40.4443.82Density1200012000Seed cotton yield（kg）364.06389.40lint cotton yield（kg）147.22170.6

26、4Increasing rate（%）-15.90单铃重VdAL foliage spraying increase cotton yield（2015-11，Xinjiang，8ha）（叶面喷施棉花增产）5.255.35.355.45.455.55.55CK1/330034363840424446CK1/3300衣分衣分5.455.55.555.65.655.75.755.85.855.95.95CK1/3300单株成铃VdAL foliage spraying increases yield of lettuce（2017-9，Zhangjiakou，Heibei，1.3ha）（叶面喷施结

27、球生菜增产）CKVdALGross weight/5 plantsNet weight/5plantsDiameter(cm）Commodity rate（%）Sugar degree（%）number per unit area（mu）Gross Yield（mu）Net Yield（mu）1/60005.4223.13513.93953.94474.54852.152805.51CK4.2472.69611.61853.64003.53400.572158.69Increasing rate27.67%16.28%19.98%11.76%8.33%42.69%29.96%VdAL foli

28、age spraying increases yield of pepper（2018，Anhui）（叶面喷施辣椒增产）VdAL foliage spraying increases yield and improve quality of pepper（2018，Tongzhou，Beijing）（叶面喷施辣椒增产、改善品质）VdAL foliage spraying increases yield of tomato（2017，Zhangjiakou，Hebei）（叶面喷施番茄增产）叶 色 果实大小均匀度 挂果数量 长势VdAL foliage spraying increases ric

29、e yield（叶面喷施水稻增产）（2015-10，China Agricultural University，Beijing，Shangzhuang）Compared with the control,the grain number per panicle,1000-grain weight and rice yield of 1/10000 VdAL treatment increased by 12.28%.02230230240240250250CKCK1/100001/10000 1/200001/20000 1/400001/40000

30、Grain number/panicle28.528.5292929.529.5303030.530.5313131.531.5CKCK1/100001/100001/200001/200001/400001/400001000-grain weight0 0500500002500250030003000CKCK1/100001/100001/200001/200001/400001/40000Total dry weight（g）0 0020003000300040004000500050006000600070007000

31、80008000CKCK1/100001/100001/200001/200001/400001/40000Yield（kg/ha）CKVdAL1/10000VdAL foliage spraying increase rice yield（2018，Xiangyin，Hunan）湘早籼24号（叶面喷施水稻增产）VdAL promotes maturation and improve quality（2017-9，Zhuozhou，Heibei，Chinese crabapple）（促早熟）VdALck5000 times VdAL diluent significantly promoted

32、 early ripening,and fruit coloring period earlier.VdALckVdAL promotes maturation of Chinese date（2017-8，Xinjiang，2.7ha）（促早熟）Spraying at flowering stage:the color change is significantly faster than the control,and the taste is more crisp and sweet.CKVdALVdAL improve sweet potato quality（2018，Handan，

33、Hebei）（改善甘薯品质）VdAL foliage spraying improve watermelon quality（2018，Yanqing，Beijing）（改善西瓜品质）National test and demonstration of rice in 2018（2018年水稻全国试验示范）VdAL可促进水稻壮苗、早发、抗病、增产。增产幅度5.5-41.43%，稳定增产10%左右。VdAL regulates the expression of many genes（调控基因表达）photosyntheticVdAL spraying for 24 hours signific

34、antly affected plant gene differential expression,especially in plant hormone signal transduction,photosynthetic and circadian rhythm.VdAL regulates the expression of many genes（调控基因表达）Disease-resistant signalGlycometabolismHormone signal transductionThe proposed model for VdAL（作用机理）TFsAAAAAAAAAAAAV

35、dALTFsAAAAAAAAAAAAAAAAAATFsAAAAAAAAAAAAAAAAAAVdALVdALVdALVdALVdALVdALLow concentrationSuitable concentrationHigh concentrationAAAAAAPromote growthStrong DefenseSmall responseVdAL is supposed to be perceived by receptor-like protein kinases that would tell the plant cells to prepare for defense by ef

36、ficiently increasing photosynthesis and nutrient usage at suitable concentrations.However,at high concentration,plants will consume more energy for defensing this protein,which may increase the disease resistance,but influence the growth.VdALA novel protein stimulant（总结）1.VdAL can be produced by GES

37、 fermentation and the quality can be easily controlled,the spray-dry powder products are very stable.2.VdAL can activate various phytohormone pathways and PTI as well as ETI increase innate immunity responses and photosynthesis,and modulate the metabolism of sugar and starch.3.VdAL can increase the

38、biomass or yield in various vegetables and crops,and improve the nutrients and quality of various fruits.4.VdAL is friendly to environment and does not produce any harmful residues in nature.VdAL is very easy to use and the usage amount is very low.5.So we hope the miracle protein will help the hungry world!Cui yinzhong: http:/ are open to co-operations on all crops,vegetables and fruits.Thanks for your attention!VdALNo VdAL