自然衰老棉花种子的生理变化及ATP合成酶亚基mRNA的完整性

doi:10.3864/j.issn.0578-1752.2023.10.001

Abstract

Abstract:

【Objective】Seed aging is a complex biological process, previous studies have been used to elucidate the events. However, the mechanism of seed aging is still unclear. The naturally aged cotton seeds were used as experimental materials, and the physiological and biochemical changes as well as the changes in ATP synthase mRNA integrity that occurred in cotton seed during storage were investigated in order to provide a foundation for further illuminating the aging mechanism of cotton seeds.【Method】In this study, a collection of seeds (cultivar Xinluzao 74) that had been stored for 3 and 5 years served as the experimental materials, the newly harvested seeds were used as the control (CK). The germination percentage, water absorption and viability of cotton seeds were valued by germination test between paper, low constant temperature over method, and TTC staining method, respectively; The acid value and respiratory rate of cotton seeds were determined by the acid-base titration method, and the ATP synthase activity was detected with plant ATP synthase ELISA Kit. The mRNA integrity of ATP synthase subunit α, β, γ, ε, and δ in cotton embryo was analyzed by reverse transcription blocking-double primer amplification method.【Result】Our data suggest that seed vigor dramatically decreased over storage time. After 3 and 5 years of storage, the germination percentage of cotton seeds was significantly decreased from 98.7% to 84.0% and 58.0%, respectively (P<0.05). At the initial stage of seed imbibition (the first 4 h), the water absorption rate of seeds was significantly decreased by 11.0% and 26.9%, respectively. The results of TTC staining showed that only the radicle was slightly stained in seeds preserved 5 years but not the cotyledons and other organs stained; The acid value of seeds was significantly increased by 28.4% and 40.0%, respectively (P<0.05), this indicated that severe hydrolysis of lipid occurred in seeds. Seed respiration rate and ATP synthase activity showed an increasing trend during imbibition, but the increasement was significantly decreased (P<0.05); The respiration rate of seeds was reduced by 33.3% and 49.2% after 24 hours of imbibition, and the activity of ATP synthase was decreased by 17.9% and 73.4% after 12 hours of imbibition, respectively. The results of reverse transcription blocking-double primer amplification showed that the R value of ATP synthase subunits α, β, γ, and δ mRNAs stored in seeds were significantly decreased, but the subunit ε mRNA was significantly increased. These results indicated that the integrity of the ATP synthase subunits mRNA decreased to varying degrees during the natural storage process.【Conclusion】These results showed that a prolonged storage time could reduce seed vigor; The integrity loss of ATP synthase subunit mRNAs stored in seed embryos would cause ATP synthase subunit to be impaired and ATP synthase activity declined, thus lead to a decreased production of ATP and affect seed germination capacity. This might be one of the important reasons for cotton seed aging.

Key words: cotton seed, seed senescence, seed vigor, ATP synthase, mRNA integrity

SONG Ci, GU FengXu, XING ZhenZhen, ZHANG JunMing, HE WenXue, WANG TianBo, WANG YuLu, CHEN JunYing. Physiological Changes and Integrity of ATP Synthase Subunits mRNA in Naturally Aged Cotton Seeds[J].Scientia Agricultura Sinica, 2023, 56(10): 1827-1837.

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References 48

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ATP合成酶亚基 ATP synthase subunits	基因 Gene	正/反向引物序列 Forward/Reverse primes sequence (5′-3′)	引物长度 Primer length (bp)	退火温度 Annealing temperature (℃)	产物大小 Product size (bp)
α	XM_016819324-5	F：GCACTGGATCTATTGTGG	18	58.9	108
	XM_016819324-5	R：GTTCGTGATCGCTTAGAG	18	59.0	108
	XM_016819324-3	F：AACACGAACTCTATCGGA	18	59.1	105
	XM_016819324-3	R：TGTGCCTTTCTATTAAGCC	19	59.0	105
β	XM_016824591-5	F：AAGATCACTGATGAGTTCAC	20	58.9	199
	XM_016824591-5	R：CATCCATAGCAATGGTCC	18	59.0	199
	XM_016824591-3	F：CGAGCTTAGTGAAGATGATAA	21	59.1	128
	XM_016824591-3	R：TCTCCTTCAACTCCACAT	18	58.9	128
γ	XM_016875018-5	F：TGATACACGAACAACAGC	18	59.1	187
	XM_016875018-5	R：ATATAACGAACTCCAATAGGAC	22	59.0	187
	XM_016875018-3	F：TGTTCGGACTTGTTCATG	18	59.0	136
	XM_016875018-3	R：TACCAACTTATGCTGATGTC	20	59.0	136
ε	XM_016862594-5	F：ACAACACAGCCAAAGAAA	18	59.1	97
	XM_016862594-5	R：GCATATGTTCGAGTAGGTTAT	21	59.1	97
	XM_016862594-3	F：ATGATGTTGCACATCCTAAT	20	59.0	91
	XM_016862594-3	R：ACAAGAATACTTGATAACCCAT	22	59.1	91
δ	XM_016861180-5	F：AACATGGACCCTCCTAAA	18	58.9	88
	XM_016861180-5	R：AATTGACAGTAAGCTTGGAA	20	59.1	88
	XM_016861180-3	F：CTTAAGATTCATATGATGGATGC	23	59.1	198
	XM_016861180-3	R：TTGAACTGCAATAAGAACAAC	21	59.1	198
内参 Internal reference	NM_001155179.1(Actin)-5	F：GTATGAGCAAGGAGATCAC	19	58.9	194
内参 Internal reference	NM_001155179.1(Actin)-3	R：TTAGAAGCACTTCATGTGG	19	59.0	194

Physiological Changes and Integrity of ATP Synthase Subunits mRNA in Naturally Aged Cotton Seeds

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