Team:Kyoto/Workflow

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Table of contents

• 1) Creation of KO yeast strains
• 2) Plasmid construction
• 3) Assesment of halotorelance
• 4) Assesment of the amount of absorbing Na+
• 5) Assesment of aggregation

1)Creation of KO yeast strains

At a first step, we worked on creation of KO yeast strains which uptake more sodium and therefore show salt-sensitivity. It is because we need to use salt sensitive yeast strains in order to assess functions of proteins which contribute salt torelance, and also Na+を外に漏らさずため込ませたかったからだ。 作り方はSpecial protocolを見てください。 Based on Aachen 2017's result, we created ΔNHA1, ΔENA1ΔNHA1,ΔENA1 at first, and 実験を進めるにあたって東北大学の魚住先生にいただいたG19株(ΔENA1,2,3,4)がよく塩を吸収し、高い塩感受性を示すことがわかり、ENA1だけでなく同じタンデムにあるENA2,3,4もノックアウトする方がいいことがわかった。またNHA1をノックアウトすることも塩吸収に貢献していたため、最後にΔENA1,2,5ΔNHA1も作成しました。以下が私たちが作成した変異株です。実験においては、上記のG19株と、渡部先生にいただいた醤油酵母も用いた。

• ・ΔNHA1
• ・ΔENA1ΔNHA1
• ・ΔENA1
• ・ΔENA1,2,5ΔNHA1

2)Plasmid construction

次に、私たちはプラスミドのコンストラクションを行いました。デザインページにあるように、塩耐性のためにMangrin, ZrGPD1,ZrFPS1を、塩の回収のためにAtHKT1,AVP1, AtNHXS1, SseNHX1の作成をしました。

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3)Assesment of halotorelance

4)Assesment of the amount of absorbing Na+

5)Assesment of aggregation

Figure 2-a caption caption caption caption caption

Reference

• [1] X. rong Wang, X. Cheng, Y. dong Li, J. ai Zhang, Z. fen Zhang, and H. rong Wu, “Cloning arginine kinase gene and its RNAi in Bursaphelenchus xylophilus causing pine wilt disease,” Eur. J. Plant Pathol., vol. 134, no. 3, pp. 521–532, 2012.
• [2] A. Sigova, N. Rhind, and P. D. Zamore, “A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe,” genes Dev., 2004.
• [3] R. Esteban and R. B. Wickner, “A new non-mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains.,” Genetics, 1987.
• [4] M. T. B. Sloan, Katherine E, Pierre-Emmanuel Gleizes, “Nucleocytoplasmic Transport of RNAs and RNA–Protein Complexes,” J. Mol. Biol., vol. 428, no. 10, pp. 2040–2059, 2016.
• [5] V. W. Pollard and M. H. Malim, “the Hiv-1 Rev Protein,” Annu. Rev. Microbiol., vol. 52, no. 1, pp. 491–532, 1998.