Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant

碩士 === 國立中興大學 === 生物科技學研究所 === 104 === A T-DNA mutant, named as Large Spikelet (LS1), was noticed previously due to its increased length, by 19%, and width, by 15%, in grains, compared to the wild type rice (TNG67). The selfed progenies LS1-3, LS1-4 and LS1-5 still produced heavier grains than TNG67...

Full description

Bibliographic Details
Main Authors: Wen-Shan Tai, 戴玟姍
Other Authors: 呂維茗
Format: Others
Language:zh-TW
Published: 2016
Online Access:http://ndltd.ncl.edu.tw/handle/74165081522937928007
id ndltd-TW-104NCHU5111022
record_format oai_dc
spelling ndltd-TW-104NCHU51110222017-01-06T04:19:41Z http://ndltd.ncl.edu.tw/handle/74165081522937928007 Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant 造成大穀粒形成的基因之選殖與功能研究 Wen-Shan Tai 戴玟姍 碩士 國立中興大學 生物科技學研究所 104 A T-DNA mutant, named as Large Spikelet (LS1), was noticed previously due to its increased length, by 19%, and width, by 15%, in grains, compared to the wild type rice (TNG67). The selfed progenies LS1-3, LS1-4 and LS1-5 still produced heavier grains than TNG67, though the fertility rate of LS1 was severely decreased to 3~6%. In order to investigate the molecular mechanism of LS1, inverse PCR was performed and the result indicates an integration site of T-DNA located between the Hemolysin-III (HlyIII) and Sucrose Phosphate Synthase (SPS) genes. Genomic PCR on LS1 and its progenies further confirmed the result. Unfortunately, no segregation line without T-DNA insertion was identified among the tested progenies; the genetic linkage between the T-DNA insertion and the Large Spikelet phenotype was therefore un-confirmed. SPS encodes a key enzyme involved in starch and sucrose metabolism. As T-DNA is located downstream of the SPS gene, the expressions of SPS was unchanged in LS1 according to Real-time PCR quantitation. This result rules-out the possible role of SPS in LS1 phenotype. HlyIII encodes an integral membrane receptors, belonging to the Progestin and AdipoQ Receptors (PAQR) family, with seven transmembrane domains. Disruption of HlyIII in Arabidopsis renders plants with hypersensitivity to ABA/osmotic stress; therefore, HlyIII may play a negative role in ABA/osmotic signaling. Rice has six PAQR members. As HlyIII share at most 58% similarity in protein sequence to the other members, HlyIII may play a different function from its kin in rice. According to transcriptome information from the TIGR databank, HlyIII was highly expressed in the endosperm 25 days after pollination. Moreover, expression of HlyIII was higher in LS1 and its selfed progenies than in the TNG67, suggesting an effect of T-DNA insertion. We suspected that over expressions of HlyIII may be responsible the LS1 phenotype. However, further examinations using the HlyIII-overexpressed and HlyIII-silenced transgenic rice, together with the HlyIII T-DNA-tagged Arabidopsis, did not correlate the LS1 phenotype with the HlyIII gene expression or gene integrity. HlyIII was also known to play an important role in phosphate metabolism in Saccharomyces cerevisiae; therefore, we compared the phosphate content between LS1 and TNG67 plants. Surprisingly, both LS1 and its selfed progenies exhibited higher phosphate content than TNG67, especially in the leaf sheath. Unfortunately, the correlation between the HlyIII gene and phosphate content was not found in the HlyIII transgenic plants. In conclusion, the Large Spikelet phenotype is not related to the HlyIII gene but may be caused by somatic mutations that happened accidently during the previous transformation process of TNG67. 呂維茗 2016 學位論文 ; thesis 54 zh-TW
collection NDLTD
language zh-TW
format Others
sources NDLTD
description 碩士 === 國立中興大學 === 生物科技學研究所 === 104 === A T-DNA mutant, named as Large Spikelet (LS1), was noticed previously due to its increased length, by 19%, and width, by 15%, in grains, compared to the wild type rice (TNG67). The selfed progenies LS1-3, LS1-4 and LS1-5 still produced heavier grains than TNG67, though the fertility rate of LS1 was severely decreased to 3~6%. In order to investigate the molecular mechanism of LS1, inverse PCR was performed and the result indicates an integration site of T-DNA located between the Hemolysin-III (HlyIII) and Sucrose Phosphate Synthase (SPS) genes. Genomic PCR on LS1 and its progenies further confirmed the result. Unfortunately, no segregation line without T-DNA insertion was identified among the tested progenies; the genetic linkage between the T-DNA insertion and the Large Spikelet phenotype was therefore un-confirmed. SPS encodes a key enzyme involved in starch and sucrose metabolism. As T-DNA is located downstream of the SPS gene, the expressions of SPS was unchanged in LS1 according to Real-time PCR quantitation. This result rules-out the possible role of SPS in LS1 phenotype. HlyIII encodes an integral membrane receptors, belonging to the Progestin and AdipoQ Receptors (PAQR) family, with seven transmembrane domains. Disruption of HlyIII in Arabidopsis renders plants with hypersensitivity to ABA/osmotic stress; therefore, HlyIII may play a negative role in ABA/osmotic signaling. Rice has six PAQR members. As HlyIII share at most 58% similarity in protein sequence to the other members, HlyIII may play a different function from its kin in rice. According to transcriptome information from the TIGR databank, HlyIII was highly expressed in the endosperm 25 days after pollination. Moreover, expression of HlyIII was higher in LS1 and its selfed progenies than in the TNG67, suggesting an effect of T-DNA insertion. We suspected that over expressions of HlyIII may be responsible the LS1 phenotype. However, further examinations using the HlyIII-overexpressed and HlyIII-silenced transgenic rice, together with the HlyIII T-DNA-tagged Arabidopsis, did not correlate the LS1 phenotype with the HlyIII gene expression or gene integrity. HlyIII was also known to play an important role in phosphate metabolism in Saccharomyces cerevisiae; therefore, we compared the phosphate content between LS1 and TNG67 plants. Surprisingly, both LS1 and its selfed progenies exhibited higher phosphate content than TNG67, especially in the leaf sheath. Unfortunately, the correlation between the HlyIII gene and phosphate content was not found in the HlyIII transgenic plants. In conclusion, the Large Spikelet phenotype is not related to the HlyIII gene but may be caused by somatic mutations that happened accidently during the previous transformation process of TNG67.
author2 呂維茗
author_facet 呂維茗
Wen-Shan Tai
戴玟姍
author Wen-Shan Tai
戴玟姍
spellingShingle Wen-Shan Tai
戴玟姍
Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant
author_sort Wen-Shan Tai
title Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant
title_short Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant
title_full Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant
title_fullStr Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant
title_full_unstemmed Molecular Characterization and Functional Study of a Large Spikelet (LS1) Rice Mutant
title_sort molecular characterization and functional study of a large spikelet (ls1) rice mutant
publishDate 2016
url http://ndltd.ncl.edu.tw/handle/74165081522937928007
work_keys_str_mv AT wenshantai molecularcharacterizationandfunctionalstudyofalargespikeletls1ricemutant
AT dàiwénshān molecularcharacterizationandfunctionalstudyofalargespikeletls1ricemutant
AT wenshantai zàochéngdàgǔlìxíngchéngdejīyīnzhīxuǎnzhíyǔgōngnéngyánjiū
AT dàiwénshān zàochéngdàgǔlìxíngchéngdejīyīnzhīxuǎnzhíyǔgōngnéngyánjiū
_version_ 1718406513646632960