Bananas 1 - Aminocyclopropane-1 - Carboxylic Acid Synthase (acs) Antisense Gene Expression Vector Construction And Transformation Of Banana Research

PedF:wtpe9n of tb. Popetruct with antiseuse cDNA encoding'.1 opiPqcyd9p rOP.PP 1 -carbo xylot sy uthase to b a n a na. j..(Musa spP. M^ ~roup)(Mus. spp. M^ ~roup)Abstracti i1w:, lt;.' Tissue specific exPression of ACS cDNA. We used Northem narrowFpsct h!9t hybridiZaion to apalyze the ACS cDNA exPression in differenttissues (leff rPot, peel gnd pulp) of banana with the 3' RACE produc aspr9be. The hybri4ization result suggested that this ACS cDNA expresSedespecially in banana ffoit. Comparing the nucleotides sequence and deducedamino aci4s seqqope of this ACS cDNA with these in Genebank shows99.$)/o 4opeOlogy in Puclebtid6s sequence and high identity in amino acidssequ?nce of MA-ACSl. The ACS encoded by MA-ACSl carelyses AdoMetinto ACC during banana fruit ripening.9: obtaining the fyJt length of the cDNA encodingl - aminocyc l opropane- l -carboxghe,inthtwS ). TWO fragmentS of thecDNA encoding ACS in banana fruit have been obtained byvopR. OneJ'; tfmpePt with the intact 5' end,of ACS cDNA obtained by using raPidpsplifiFaiOn of cDNA 5' end (5' RACE) was cloned in pGEM T-EASYvector, and the other with the intact 3' end of ACS cDNA obtained by using\. .. ?: '!,,3! %qB was cloned in pCR2. 1vector respectively. The overlap betheen thetyo.fragments was removed with MfeI restriction site. The 5' RACE productexcised from pGEM T-EASY vector with EcoRI was cloned intO pBluscriptMl3.and.this newly formed construct was designaed pBS-667. Subsequentlythe 3, nyCE PrOduct excised from pCR2.1 vector with Mfel/XbaI sites wasinSerted into pB5f667 and the construct was designated p8S-ACC because..: (.\'. 1e t': 1\. -the conStruCt contained the full lengh of the ACS cDNA.i;\ 1.,; t.,.;..t ty. construPion of intermediate expression vector with the antisense^CS FPNA, Th9 full length of ACS cDNA excised hom PBS-ACC withSPryb4I sites was inserted into plasmid pBBB digested with SalimbaI inord9r of 35S promoter - 3' ACS cDNA 5' - boinatoL The newlyconstruct with anisense ACS cDNA was resulted in. The electrophoresis ofthe construCt digested with SalI/XbaI showed .that the intermediate expressionvector was obtained and the construct was na!ned as pBBB-aACC.- 73 -?Genetic transformation of banana. Apical meristems and corms from shoots of tissue cultured banana plantlets were used as material in genetic transformation via agrobacterium-mediated or microprojetile bombardment. Among the transformed buds, there are 64 regenerated plants resistant to PPT. Among the regenerated plants, there are 9 putative transgenic plants have been checked by using PCR.