阅读说明：本技术 一种植物叶片衰老指数的测定方法 (A kind of measuring method of plant leaf blade senescence index ) 是由 黄升谋 于 2018-05-21 设计创作，主要内容包括：本发明提供一种植物叶片衰老指数的测定方法。该方法如下：首先提取植物叶片的总RNA,以甲醛-琼脂糖凝胶电泳法对其进行电泳,凝胶成像系统照相,分析总RNA的提取质量。用紫外可见分光光度计测定其含量和纯度。然后分离纯化mRNA,用紫外可见分光光度计测定其含量和纯度。用mRNA含量/总RNA含量表示叶片衰老指数。该方法灵敏、准确,反映了叶片衰老的本质。(The present invention provides a kind of measuring method of plant leaf blade senescence index.This method is as follows: extracting the total serum IgE of plant leaf blade first, carries out electrophoresis to it with formaldehyde-agarose gel electrophoresis, the extraction quality of total serum IgE is analyzed in gel imaging system photograph.Its content and purity are measured with ultraviolet-uisible spectrophotometer.Then mRNA is isolated and purified, measures its content and purity with ultraviolet-uisible spectrophotometer.Leaf senile index is indicated with mRNA content/total RNA content.This method is sensitive, accurate, reflects the essence of leaf senile.)

1. total RNA content is used for the measurement of the index as leaf senile, its step are as follows:

1, it takes 100mg sample to be put into liquid nitrogen to grind and be homogenized；

2, the guanidine radicals sulphur hydracid buffer of 450ul, and vortex oscillation is added, makes nuclease deactivation, to obtain complete core Acid；

3, sample liquid is moved into QIAshredder column spinner, with 14000rpm centrifugation 2 minutes at 20 DEG C；

4, the ethyl alcohol of 225ul is added in supernatant, the condition of pellosil constraint nucleic acid is provided；

5, sample is added in the RNeasy mini column spinner containing pellosil, total serum IgE is adsorbed on pellosil, with buffering Various pollutants are gone in liquid elution, and total serum IgE is eluted from RNeasy mini column spinner with the water of deoxyribonuclease；

6, electrophoresis is carried out to it with formaldehyde-agarose gel electrophoresis, the extraction matter of total serum IgE is analyzed in gel imaging system photograph Amount；

7, its content and purity are measured with ultraviolet-uisible spectrophotometer.

2. mRNA content is used for the measurement of the index as leaf senile, its step are as follows:

1, the OBB buffer of 250ul and the oligotex suspension of 15ul are added in the total serum IgE sample of 250ul and mixes equal It is even, it is incubated 3 minutes in 70 DEG C of water-baths, room temperature is transferred 10 minutes, hybridizes the Poly-A of mRNA with oligotex particle；

2, with 18000g centrifugation 2 minutes at 20 DEG C, to precipitate oligotex-mRNA compound；

3, it with the buffer solution OW2 of 400ul again suspension oligotex-mRNA and is transferred in a small column spinner, at 20 DEG C Under with 18000g centrifugation 1 minute, repeat this step it is primary；

4, finally plus buffer OEB is in small column spinner, with 18000g centrifugation 1 minute at 20 DEG C, mRNA from oligotex- 5. are eluted in mRNA measures its content and purity with ultraviolet-uisible spectrophotometer.

3. using mRNA/total serum IgE as the index of leaf senile:

Leaf senile index calculates: leaf senile index=mRNA content/total RNA content.

Technical field: the present invention relates to a kind of measuring methods of plant leaf blade senescence index.

Technical background: in plant growth and development process, oxygen can be absorbed, generates reactive oxygen species and free radicals, oxidative cell DNA, RNA, protein and biological film phospholipid, destroy inhereditary material, make protein function decline or loss of function, make to give birth to Peroxidatic reaction of lipid occurs for the side chain of the polyunsaturated fatty acid of object membrane phospholipid, forms lipid peroxidation product (Lipid PerOxide, LPO) such as malonaldehyde (Malonaldehyde, MDA), so that the mobility of cell membrane and permeability is changed, Eucaryotic cell structure change and function reduction are eventually led to, leaf senile is caused.When plant leaf blade aging, photosynthesis declineD, seriously Influence the yield and quality of crop.

Currently, people are generally using MDA content as the index of cell or tissue aging.However, MDA it only reflect that cell is anti- The ability for aoxidizing one side, cannot reflect the physiological activity and aging aspects of cell completely.It declines so inventing a kind of plant leaf blade The measuring method of old index improves crop yield and quality is of great significance for preventing crop leaf aging.

Summary of the invention:

All variations of plant are all the intracorporal functional mass of plant --- the albumen based on the metabolism of protein and nucleic acid Matter is to be transcribed into ribonucleic acid (RNA) by DNA (DNA), made of then being translated again as RNA.Protein and RNA Synthesis reflect cell viability (aging degree), however, since there is structural proteins and storage protein, Ta Menyu into the cell Cell viability is unrelated, and therefore, the index using protein content as cell ageing is insensitive.

Total serum IgE includes rRNA, tRNA, microRNA and mRNA in core, and stability is preferable in vivo by rRNA, content It is higher, can there are long period, referred to as long-life RNA, they cannot delicately change with the variation of cell activity, and mRNA It is unable to long-term existence in plant, is degraded quickly by RNA enzyme after functioning, mRNA content reflects Gene Transcription in vitro Just.MRNA content is lower, only accounts for the 1-5% of total serum IgE, and change with the physiological status of cell, therefore mRNA/total serum IgE is more It can reflect the height of cytogene transcriptional activity.Have using mRNA/total serum IgE as the index of leaf senile higher sensitive Property.

The extracting method of total serum IgE.It first takes 100mg sample to be put into liquid nitrogen to grind and be homogenized, the rear guanidine radicals sulphur that 450ul is added Hydracid buffer, and vortex oscillation make nuclease deactivation, and to obtain complete nucleic acid, sample liquid is moved into In QIAshredder column spinner, with 14000rpm centrifugation 2 minutes at 20 DEG C, the ethyl alcohol of 225ul is added in supernatant, provides Pellosil fetters the condition of nucleic acid, sample is added in the RNeasy mini column spinner containing pellosil, total serum IgE is adsorbed onto On pellosil, various pollutants are gone with buffer elution.Finally with the water of deoxyribonuclease total serum IgE from RNeasy mini It is eluted in column spinner.Electrophoresis is carried out to it with formaldehyde-agarose gel electrophoresis, gel imaging system photograph, analysis is always The extraction quality of RNA.Its content and purity are measured with ultraviolet-uisible spectrophotometer.

The Isolation and purification method of mRNA.The OBB buffer and 15ul of 250ul are added in the total serum IgE sample of 250ul Oligotex suspension is simultaneously uniformly mixed, and is incubated 3 minutes in 70 DEG C of water-baths, and room temperature is transferred 10 minutes, makes the Poly-A of mRNA Hybridize with oligotex particle, then with 18000g centrifugation 2 minutes at 20 DEG C, to precipitate oligotex-mRNA compound.With The buffer solution OW2 of 400ul suspension oligotex-mRNA and is transferred in a small column spinner again, at 20 DEG C with 18000g is centrifuged 1 minute, and it is primary to repeat this step.Finally plus buffer OEB is in small column spinner, at 20 DEG C with 18000g from The heart 1 minute, mRNA is eluted from oligotex-mRNA.Its content and purity are measured with ultraviolet-uisible spectrophotometer.

Leaf senile index calculates: leaf senile index=mRNA content/total RNA content.