10905

10047

كارشناسي ارشد

بيوتكنولوژي كشاورزي

اصفهان: دانشگاه صنعتي اصفهان، دانشكده كشاورزي

1394

شانزده، 124ص.: مصور

مسعود بهار، مجيد طالبي

آقافخر ميرلوحي

1394/10/26

آذر شاهپيري، امير مساح

مهندسي كشاورزي

ID10047

ج بررسي بيان نسبي ژن 96 uf3gt 3 8 نتايج استخراج كل محتواي آنتوسيانين از غده هاي ارقام مختلف سيب زميني 89 3 01 مقايسه نتايج Real time PCR با غلظت آنتوسيانين و رنگ غده ها در ارقام مختلف 001 3 01 جداسازي ژن هاي كد كننده ايزوفرم هاي ژن chs در سيب زميني 201 3 01 1 واكنش PCR با دماي اتصال بهينه شده 301 3 01 2 برش آنزيمي 401 3 01 3 همسانه سازي قطعات تكثيري ايزوفرم 1 و 2 در پالسميد pTZ57R T و انجام واكنش 107 Colony PCR 3 01 5 استخراج پالسميد و انجام واكنش PCR بر روي پالسميد استخراج شده 801 3 01 6 مقايسه نتايج توالي يابي و تاييد صحت جداسازي ژن هاي كد كننده ايزوفرم هاي 1 و 2 901 فصل چهارم نتيجه گيري كلي و پيشنهادات 4 1 نتيجه گيري كلي 211 4 2 پيشنهادها 511 منابع 611 چكيده انگليسي 621 يازده

Detection and Expression Analysis of The Key Genes Involved in Anthocyanin Biosynthetic Pathway of Different Potato Cultivars Reza Lotfi rezalotfi7470@gmail com December 8 2015 Department of Agricultural Biotechnology Isfahan University of Technology Isfahan 84156 83111 IranDegree M Sc Language FarsiSupervisors M Bahar mbahar@cc iut ac ir M Talebi mtalebi@cc iut ac irAbstractPotato Solanum tuberosum as a major source of starch protein antioxidants and vitamins is one of themost nutritious crops in the world An important physiological aspect of potato is the anthocyanincompounds in its tuber Anthocyanins are protective against pests and pathogens attractive for pollinatorsand have role in signaling of plant microbe interaction The variation of anthocyanin contents in specifictissues induces the tuber color into red blue and purple Anthocyanins also reduce the risk of human cancerby their antioxidant activity to prevent the free radicals action There are two groups of genes involved inanthocyanin biosynthetic pathway in plants Of which structural genes such as chs chi ans and uf3gt codefor necessary enzymes in pigment biosynthesis process and the second group as transcription factors regulates the function of structural genes Due to lack of detailed information about the function of structuralgenes in anthocyanin biosynthetic pathway in potato plant the present research focused on detection andexpression analysis of chs chi ans and uf3gt genes in tuber skin and flesh tissue of different potato cultivarswith various tuber colors To detect the corresponding genes in the cultivars the PCR reaction wasperformed using specific primer pairs designed from conserved sequences of the genes in the NCBIdatabase The amplification of DNA bands with expected sizes in PCR reactions documented the presenceof analogous gene in tested potato cultivars The detection of anthocyanin pathway genes in potatoes wasverified by restriction enzyme analysis and nucleotide sequencing of the amplified fragments The sequenceanalysis revealed that the chs chi ans and uf3gt genes detected in the potato cultivars are homologus andsimilar to those previously sequenced and deposited in NCBI database The RNA extraction and cDNAsynthesis from the tuber skin and flesh tissues of the potatoes were done and the expression of each genewas analyzed in common RT PCR and Real time PCR reactions In the expression analysis chs ans anduf3gt genes were successfully amplified whereas chi gene failed to produce the expected fragment Basedon the results of Real time PCR reaction chs ans and uf3gt were differently expressed in tuber skin andflesh tissues of the cultivars examined In both skin and flesh tissues of the red and purple tubers theexpression level of these genes was significantly p 0 05 higher than white cultivars Accordingly theanthocyanin content of colored potatoes was significantly p 0 05 higher than white tubers In this study the presence of both isoform 1 Stchs1a Stchs1b and isoform 2 Stchs2 of chs gene was verified in skinand flesh of potato tubers using specific primer pairs The restriction enzyme analysis and nucleotidesequencing validated the specific amplification of each isoform in the tubers and the RT PCR resultsrevealed that both isoforms are expressed simultaneously to direct anthocyanin biosynthesis in coloredpotato tubers Based on the Real time PCR and anthocyanin extraction results it can be concluded that thecoloration and anthocyanin contents of potato tubers is correlated to the expression of chs ans and uf3gtgenes The results indicated that chs ans and uf3gt are the key genes in anthocyanin biosynthesis pathwayof colored potato cultivars while chi does not seem to have a crucial role in anthocyanin biosynthesisprocess Keywords Potato Anthocyanin Gene detection Gene expression analysis Real timePCR

مسعود بهار، مجيد طالبي

آقافخر ميرلوحي

آذر شاهپيري، امير مساح