Isolation of genomic DNA fragments from jasmine rice which are differentially expresessed in various tissues

Kalaya Silawisas

Isolation of genomic DNA fragments from jasmine rice which are differentially expresessed in various tissues

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1991

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1991

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Master Thesis

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eng

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application/pdf

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xiv, 110 leaves : ill.

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open access

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ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า

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Mahidol University

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Thesis (M.Sc. (Biochemistry))--Mahidol University, 1991

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Kalaya Silawisas Isolation of genomic DNA fragments from jasmine rice which are differentially expresessed in various tissues. Thesis (M.Sc. (Biochemistry))--Mahidol University, 1991. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/103284

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Isolation of genomic DNA fragments from jasmine rice which are differentially expresessed in various tissues

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การแยกจีนซึ่งแสดงออกอย่างต่างระดับในเนื้อเยื่อของข้าวไทย

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Kalaya Silawisas

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Burachai Sonthayanon
Sakol Panyim

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A genomic DNA library of Oryza sativa subspecies indica cv. KDML 105 has been constructed by using bacteriophage lambda Gem-11 as a cloning vector. DNA fragments from the rice genome were obtained by partial Sau3A I digestion followed by size-fractionation on a 10-40% linear sucrose gradient to get 15-19 Kb DNA fragments. They were then ligated to Bam HI digested, dephosphorylated, lambda arms. Concatemeric ligation products were then packaged in vitro and used to infect E. Coli, Kw 251. A probability of having cloned any DNA piece with an average size 18 kb in the library was about 0.95. About thirteen thousand plaques in this amplified library have been screened for clones which seems to be differentially expressed in sheath and leaf tissues of rice by plaque hybridization using 32P-labeled cDNAs as probes. Nine clones were initially selected and Northern blot hybridization was then used to confirm those which hybridized strongest to either RNA of sheath or leaf tissues. Finally, one single clone designated as L4 was chosen for study. The L4 clone has been subcloned into pSPL plasmid vector by using Xho I, which can separate the rice DNA from the lambda arms. There are six Xho I fragments of various sizes in this clone, 5.7 kb, 3.6 kb, 1.4 kb, 1.3 kb, 1.1 kb, and 0.2 kb. Relative arrangement of those Xho I fragments in L4 were later determined. It was found that their order from the right arm was 1.1 kb, 5.7 kb, 1.3 kb, 0.2 kb, 3.6 kb, and 1.4 kb fragments, respectively. It was also found that the 3.6 kb, 5.7 kb, and 1.3 kb Xho I fragment s have their RNA counterparts whereas 1.4 kb and 1.1 kb do not. The 3.6 kb xho I fragment gives different hybridization patterns with RNA from sheath and leaf tissues of rice on Northern blot analyses but 5.7 kb and 1.3 kb Xho I fragments give another unique pattern. Data available indicate that there may be two small genes of rice lying in the L4 clone, on in the 5.7 kb and 1.3 kb fragments and the other in the 3.6 kb fragment. The two genes, however, do not have DNA sequence identity. The putative gene contained in the 3.6 kb DNA fragment may be part of a multi-gene family since the DNA fragment appeared to be present in multiple copies in the rice genome.