CAJ | 학술논문

为了将斑鸠菊( Vernonia galamensis)二酰甘油酰基转移酶基因( V gDGA T1a)导入棉花,创制转基因高油棉花种质,优化建立农杆菌介导的棉花茎尖转化体系,以中棉所49茎尖为外植体,以 H ptⅡ基因为筛选标记, V gDGA T1a为目的基因,用农杆菌介导法,研究外植体培养时间、浸菌侵染时间、共培养时间及吸光度 A600值等对棉花茎尖转化的影响.结果表明,在外植体培养3~5 d、生长点纵切、菌液吸光度 A600值0.6~0.9、浸菌40~60 min、浸菌后暗培养1 d和使用MSB+活性炭1 g/L+头孢霉素400 mg/L作为生根培养基的条件下能高效获得再生转化植株,从而为转基因高油棉花种质的创制提供了一种有效的方法.
위료장반구국( Vernonia galamensis)이선감유선기전이매기인( V gDGA T1a)도입면화,창제전기인고유면화충질,우화건립농간균개도적면화경첨전화체계,이중면소49경첨위외식체,이 H ptⅡ기인위사선표기, V gDGA T1a위목적기인,용농간균개도법,연구외식체배양시간、침균침염시간、공배양시간급흡광도 A600치등대면화경첨전화적영향.결과표명,재외식체배양3~5 d、생장점종절、균액흡광도 A600치0.6~0.9、침균40~60 min、침균후암배양1 d화사용MSB+활성탄1 g/L+두포매소400 mg/L작위생근배양기적조건하능고효획득재생전화식주,종이위전기인고유면화충질적창제제공료일충유효적방법.
@@@@In past two decades , abundant researches have been published on transformation , regeneration , and genetic enhancement of cotton , especially Gossypium hirsutum . Generally , genes conferring agronomic advantages have been introduced into plants via A grobacterium‐mediation or particle bombardment , and then the transgenic plants are regenerated through somatic embryogenesis from callus . However , the embryogenic callus‐based regeneration is difficult and time‐consuming in cotton . Therefore , it is necessary to establish an effective system for the A grobacterium‐mediated genetic transformation of shoot tip in cotton . @@@@Triacylglycerols ( TAG) are a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bones . Diacylglycerol acyltransferase ( DGAT ; EC3 .2 .1 .20) catalyzes the last step of TAG biosynthesis and it is the only key enzyme evolved in . DGA T1 and DGA T2 , as two types of DGA T in eukaryotes , belong to different gene families . And previous studies have reported that the expression of DGA T1 increased seed oil content and mass . @@@@In order to get new cotton germplasm with high oil content , we used the shoot tips of Zhongmiansuo 49 as explants and introduced an improved vector carrying a selection marker H ptⅡ gene and a target V gDGA T1a gene into cotton via A grobacterium‐mediated transformation . This improved A grobacterium‐mediated transformation and regeneration system were established by optimizing different parameters such as pre‐culture period of seeds , concentration of A grobacterium in solution , immersing time , co‐cultivation period and components of MSB [ Murashige and Skoog ( MS) medium + vitamins of Gamborg s (B5) medium] . @@@@Cotton seeds ( Gossypium hirsutum L . cv . Zhongmiansuo 49 ) were decorticated manually and surface sterilized in 0 .1% HgCl for 8 min , followed by rinsing with sterile distilled water for five times . The cotyledons were removed from 3‐day‐old to 5‐day‐old in vitro germinated seedlings and the shoot tips were cut in lengthwise to decrease the damage of meristematic cells . The strain EHA 105 was grown overnight on a shaker at 200 r/min and at 28 ℃ until the A600 value of bacterial concentration reached 0 .6 0 .9 . The suspension cells were centrifuged at 5 000 r/min for 10 min and the pellets were resuspended in an equivalent volume of liquid co‐cultivation medium [ MSB +200 μmol/L acetosyringone ( AS) ] . The treated explants were immediately immersed into prepared A grobacterium suspension containing pCAMBIA1301 , a binary vector carrying the V gDGA T1a gene , for 40 60 minutes . The tips were blotted dry on sterile filter paper and transferred into the co‐cultivation solid medium at 28 ℃ in dark . After co‐cultivation for 1 day , shoot tips were transferred into root induction medium containing MSB , 1 g/L activated charcoal , 400 mg/L cephalothin (Cef) and solidified with 0 .2% ( W/V ) phytagel , then grew in a growth chamber with stringent light , temperature and humidity control . In the following 3 4 weeks , 5‐leaf‐plants were transferred to plastic pots containing soil matrixes ( 1∶1 of turf and vermiculite) . Besides the optimized genetic transformation protocol of cotton shoot tip as stated above , it is found that 50 mg/L hygromycin ( Hyg) could accurately distinguish the resistant plants . In addition , reduplicated selections improved accuracy . @@@@In conclusion , this study describes an optimized transformation protocol for shoot tip of Zhongmiansuo 49 with an A . tume f aciens strain EHA105 harboring DGA T1 gene , and proves that it is an efficient and economical method to obtain transgenic plants based on the results of different and important parameters influencing the transformation efficiency .