抗黄瓜花叶病毒RNAi载体的构建及烟草的转化(3)

发布时间:2021-06-05

ZHANGYuetal.ConstructionofRNAiVectorWhichResistCucumberMosaicVirusandTransformationofTobacco71

thefragmentcontainedPCRCR2BKandPCRCR2SBwasdigestedfrompiRDCR2andconnectedtothepBi35SG12togain

(11248bp),andthefragthepBi35SCR2expressionvector

mentsof1409and9839bpcouldbeobtainedbyBamHingleⅠs

,therefore,thepBi35SCR2expressionvectorenzymedigestion

wassuccessfullyconstructed(Fig.6).

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16

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18

19

10

11

12

13

14

Table3 PercentageofnucleotideandaminoacidsequenceidentitiesofCMV2ageneofCMVisolations

AJ27648075.298.192.697.093.888.994.475.293.098.599.196.797.893.693.193.986.293.2

AB17684871.975.674.775.075.674.075.797.675.075.775.875.375.475.475.175.071.374.9AB36850096.971.992.697.293.988.794.475.693.098.798.496.898.093.893.193.686.393.6FJ26874591.272.290.791.795.688.495.974.694.893.093.091.792.595.198.195.585.595.1AB07989096.771.896.590.693.189.094.075.192.497.497.498.597.493.692.293.386.792.9AB17976592.372.092.093.691.788.998.575.097.393.994.093.094.096.596.397.686.296.3EU72357088.571.288.087.788.187.989.573.988.389.489.389.189.089.489.088.994.888.8S7218792.172.192.093.892.097.187.975.198.494.694.693.994.597.396.698.086.697.2EU66500171.897.971.872.371.971.871.371.774.475.775.875.475.274.775.074.971.374.6D8633091.771.691.493.491.396.887.598.871.493.293.292.393.195.895.596.985.495.8D1020997.572.697.291.196.992.388.392.472.491.898.897.398.794.393.393.886.993.9AB36849798.372.296.891.196.691.988.291.872.091.397.697.298.194.093.294.286.793.6EF20259695.972.095.790.598.491.488.191.672.091.096.296.097.293.592.293.286.893.0AM18311897.272.396.991.196.992.388.492.472.191.898.197.196.194.092.993.686.593.6AM18311592.372.291.693.391.494.187.994.371.993.791.891.891.192.195.396.086.495.4EF21302490.972.190.995.990.693.186.893.271.892.890.990.790.491.192.396.486.195.9DQ41273191.871.991.493.391.496.887.696.771.796.391.991.591.192.094.093.186.296.5DQ39954988.371.987.887.588.088.096.588.071.987.588.388.188.288.287.987.087.686.0

7.5NS04CMV2a91.972.091.693.691.694.887.595.271.894.791.991.491.492.193.493.495.08

Thedownleftisnucleotideidentities,theupperrightisaminoacididentities.1-19arecorrespondingwiththesequenceNo.intheleftoffigurefromuptobottom

M:DL2000;1:CR2BKsequence.Fig.3 PCRamplificationofCR2B

M:DL4500;1:piRDCR2.SwithClaI.

Fig.5 Therestrictionenzymedigestionofrecombinantplas

midpiRDCR2.

M:DL2000;1:CR2BKsequence.

Fig.4 PCRamplificationofCR2SB

ObtainingoftransgenictobaccoplantsTransgenictobaccoplantsDNAwasextracted,andthentheamplificationwascarriedoutbyprimersCR2Kp/CR2SB

,the1.2%ofagarosegelandCR2Bg/CR2SBrespectively

,itcouldbeconcludedelectrophoresiswasusedforanalysis

thatthePCR_CR2_BKfragmentwith693bpandthePCR_CR2_SBfragmentwith741bpwereamplified(Fig.7,8)

M:DL4500;1:pBi35SCR2withBamHⅠ.

Fig.6 Therestrictionenzymedigestionofrecombinantplas

midpBi35SCR2

indicatingthattheobtainedtobaccoplantshadbeensuccess

fullytransformedintothepBi35SCR2expressionvectorAgrobacteriummediatedmethod.

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