Effects Of M.Thermophilus Ferredoxin On Photosynthetic Efficiency In Rice:Normal Vs Stressed Growth Condition

Rice?Oryza sativa L.?is an important food crop in China and even in the world.Its yield has always been one of the hot issues of concern.Abiotic stresses are becoming more and more serious in recent years,which seriously affect the growth and yield of plants.Finding,screening and identifying related resistance genes and using genetic engineering to improve rice resistance are important ways to improve rice resistance and yield.The ancient bacteria live in the extreme environment,which contains many genes which are not contained in rice,including high/low PH,high salinity,and so on.If these precious genetic resources can be applied to the important grain crops in our country,it will bring revolutionary breakthrough to the conventional resistance breeding of the crops.In the early stage of this experiment,after the treatment of the thermophilic autotrophic methantobacilli?Archaea model?by abiotic stress?high temperature,high salt?,the highly expressed ferredoixn?Fd?amino acid sequence was screened by its proteomics iTRAQ analysis.And the high expression of ferredoixn?Fd?amino acid sequence was screened by mass spectrometry.In this paper,we obtained the high expression of MtFd gene in Rice by gene synthesis,and constructed the rice transformation carrier.The Agrobacterium mediated method was transferred into rice ZH 11.The difference of photosynthesis and biomass of transgenic rice was analyzed,and the resistance of transgenic rice to high salt was detected.The main results of this study are as follows:1.Construction of MtFd Overexpression Vector and Obtainment of Transgenic Rice?1?according to the amino acid sequence of thermophilic autotrophic methanotrophin,the MtFd DNA sequence was redesigned through the analysis of the preferred codon of rice,and the gene was synthesized by the company.Using transgenic vector pCAMBIA1301,successfully constructed the rice transformation vector pCAMBIA1301-MtFd.?2?Using Agrobacterium-mediated method,pCAMBIA1301-MtFd was transformed into Zhonghua 11 of rice,identified by hygromycin selection and reverse transcription PCR,and 18 transgenic plants of T₀ generation MtFd were obtained.Three independent lines?MtFd-1,MtFd-2,MtFd-5?with higher expression levels were screened by semi-quantitative PCR and planted in the T₁ generation.2.Determination of Photosynthesis Rate of MtFd Transgenic Rice PlantsThe net photosynthetic rate?Pn?and stomatal conductance?Gs?were measured by the Li-6400 portable photosynthetic system at the natural temperature and9:00-11:00 in the morning.The results showed that compared with the wild type,the net photosynthetic rate?Pn?of MtFd-1,MtFd-2 and MtFd-5?Pn?in the transgenic lines increased respectively by 51.2%,24.9%and 31.2%,and the maximum increase of 59.97%was MtFd-1-3.Stomatal conductance showed a positive correlation with photosynthetic rate,and the increase was respectively 60.5%,21.2%and 28.8%.The results showed that the heterologous expression of MtFd increased the photosynthetic rate of rice under normal growth conditions.3.Measurement of agronomic traits such as plant height,grain length,grain width,and 1000-grain weight in MtFd transgenic rice plantsThe biometric methods were used to measure the agronomic traits such as plant height,grain length,grain width and 1000 grain weight of three independent strains of T1?MtFd-1,MtFd-2,MtFd-5?.The results showed that plant height,grain width and 1000 grain weight decreased significantly,while grain length increased significantly.The height of the transgenic lines MtFd-1,MtFd-2 and MtFd-5 were respectively 86.9%,89.5%and 90.4%in the wild type,and the grain width was respectively 89.3%,89.5%and 91.9%in the wild type.The 1000 grain weight were respectively 84%,86.4%and 90.9%in the wild type,and the grain length increased respectively.by 5%,5.9%and 10.9%.5.Photosynthetic characteristics of MtFd transgenic rice seedlings under abiotic stress?1?Photosynthetic characteristics of MtFd transgenic rice seedlings under low temperature stressThe results showed that the net photosynthetic rate?Pn?of MtFd transgenic lines and wild type rice decreased significantly at the early stage of stress?0²d?compared with the normal growth conditions,and the ZH11,MtFd-1-2,MtFd-2-3and MtFd-5-1 were 11.8%,13.9%,21 under normal growth conditions in the early stage of stress?0²d?..9%,12.4%.Compared with wild type,the Pn of transgenic lines decreased faster and the photosynthetic rate was lower.At the same time,compared with the wild type,the changes in stomatal conductance?Gs?,transpiration rate?Tr?and intercellular CO₂?Ci?and net photosynthetic rate?Pn?of the transgenic lines were the same,and all showed a significant decrease.In the late stress stage?2⁴d?,the net photosynthetic rate?Pn?of MtFd transgenic plants and wild type increased slightly,but not significantly.At the same time,compared with the wild type,the stomatal conductance?Gs?,transpiration rate?Tr?and intercellular CO₂?Ci?and net photosynthetic rate?Pn?of the transgenic lines were the same,and all slightly increased.?2?Photosynthetic characteristics of MtFd transgenic rice seedlings under high temperature stressThe results showed that the net photosynthetic rate?Pn?of MtFd transgenic lines and wild type rice decreased significantly in the early stage of stress?0²d?,compared with normal growth conditions,and ZH11,MtFd-1-2,MtFd-2-3 and Mt Fd-5-1 were 60.9%and 68.3%under normal growth conditions,compared with normal growth conditions in the early period of stress?0²d?.51.9%,59.4%.Compared with wild type,transgenic lines decreased rapidly,but Pn remained at a high level.However,the decline of Pn is accompanied by the decline of Gs and Ci and the rise of Tr.In the late stage of stress?2⁴d?,the net photosynthetic rate?Pn?of MtFd transgenic plants and the wild type increased slightly,but not significantly.At the same time,with the increase of Gs and the decrease of Ci and Tr,the trend of change is not significant compared with the second day treatment.?3?Photosynthetic characteristics of MtFd transgenic rice seedlings under Salt StressThe results showed that the net photosynthetic rate?Pn?of MtFd transgenic lines and wild type rice decreased significantly at the early stage of stress?0²d?,compared with normal growth conditions,and ZH11,MtFd-1-2,MtFd-2-3 and Mt Fd-5-1 were 75.4%and 65.7%under normal growth conditions,compared with normal growth conditions in the early stage of stress?0²d?.82.1%and 64.9%.Compared with wild type,transgenic lines decreased rapidly,but Pn remained at a high level.However,the decline of Pn is accompanied by the rise of Gs and Ci and the decline of Tr.In the late stage of stress?2⁴d?,the net photosynthetic rate?Pn?of MtFd transgenic plants and wild type decreased,accompanied by the decline of Gs,Ci and Tr.Under abiotic stress,the photosynthetic characteristics of MtFd transgenic rice seedlings are similar to those of wild type,but the response to the change seems to be more rapid.This suggests that MtFd may be used to improve the tolerance of plants to stress by affecting light cooperation.