The Cultivation Of Arabidopsis Mutation Accumulation Lines Under Low CO₂

Photosynthesis usually refers to the process that plants absorb light energy,synthesize CO2 and H2O into organics,such as starch,and release O2 at the same time.It is the energy source of almost all life activities and the foundation of life.The photosynthetic types of green plants include C3 photosynthetic pathway,C4 photosynthetic pathway and CAM(Crassulacean acid metabolism),among which C3 and C4 are the main photosynthetic types.The production potential of C4 plants is much higher than C3 plants.In order to make better use of the advantages of C4 photosynthesis,many studies have tried to introduce the characteristics of C4 photosynthesis into C3 crops to improve its productivity.It is believed that C4 photosynthetic pathway evolved from C3 photosynthetic pathway,and low CO2 is the key driving force of C4 photosynthetic pathway evolution.Stress-induced mutagenesis is a non-random directional mutation mechanism that adapts to evolution.It increases genetic and epigenetic mutations in cells growing in non-adapted environments to increase adaptive evolution.The underlying molecular mechanism(introduction of mutations during DNA double-strand break repair)has also been confirmed to exist in Arabidopsis,increasing the accumulation of beneficial mutations under stress conditions.Our research group previously grew the C3 model plant Arabidopsis under low CO2 conditions and found that the expression of key C4 photosynthetic pathway enzymes such as PEPC and PEPC-K in Arabidopsis is up-regulated,and the genes involved in DNA damage sensing and repair—CIPK3,UVR3,TEB,and RBB1 were upregulated by low CO2.Our group collected the T-DNA knockout lines of these four genes and obtained the homozygous quadruple mutant cipk3/teb/uvr3/rbb1(Quad mutant)by crossings.We speculate that the genome stability of the homozygous quadruple mutant decreases.Under low CO2 stress conditions,the mutation rate will increase,and more mutations will be accumulated through multiple generations,more adaptive mutations are expected to be obtained.Meanwhile,in the previous work,the transgenic maize C4-PEPC(the first key enzyme of the C4 photosynthetic Pathway)3 Arabidopsis lines have been obtained,and oePEPC/cipk3/teb/uvr3/rbb1(oePEPC/Quad mutant)Arabidopsis homozygous lines have been obtained by crossing the overexpressed PEPC lines with cipk3/teb/uvr3/rbb1(Quad mutant)mutant lines.In this thesis,all the experiment is based on the mentioned-above Arabidopsis lines.In this experiment,different types of Arabidopsis lines were cultured in 100 ppm CO2 stress environment for several multiple generations to establish mutation accumulation lines.The effects of low CO2 on the growth and development of different Arabidopsis plant types were observed.Genome resequencing was planned for the 11th generation lines to track the genetic change in this process.The main research contents and results are as follows:1.Cultivation of mutant accumulation lines in Arabidopsis under low CO2 stress(1)Two CO2 growth conditions are set in this experiment,normal CO2(380 ppm)and low CO2(100 ppm)conditions.The wild-type(Col-0),cipk3/teb/uvr3/rbb1(Quad mutant)and oePEPC/cipk3/teb/uvr3/rbb1(oePEPC/Quad mutant)Arabidopsis lines were continuously cultivated for multiple generations and phenotyped for each generation.Under both normal CO2 and low CO2 conditions,cipk3/teb/uvr3/rbb1(Quad mutant)and oePEPC/cipk3/teb/uvr3/rbb1(oePEPC/Quad mutant)lines are smaller compared to wild-type Arabidopsis Col-0 plants,suggesting that knockout of DNA damage sensing and repair-related genes reduces plants growth.(2)The physiological data of each generation of wild-type,cipk3/teb/uvr3/rbb1(Quad mutant)and oePEPC/cipk3/teb/uvr3/rbb1(oePEPC/Quad mutant)Arabidopsis lines were measured.The results showed that the PEPC enzyme activity,chlorophyll content,leaf area,petal length,width and area of the three types of plants under low CO2 stress decreased compared with those under normal CO2 conditions,indicating that low CO2 is a severe stress to Arabidopsis and inhibits the plant growth of these three lines.Under normal CO2 and low CO2 conditions,the PEPC enzyme activity of each generation of oePEPC/cipk3/teb/uvr3/rbb1(oePEPC/Quad mutant)was significantly higher than that of the same generation of wild-type Arabidopsis and cipk3/teb/uvr3/rbb1(Quad mutant),indicating that the overexpression of PEPC gene was stable in its offspring.Under normal CO2 and low CO2 conditions,the leaf area and petal area of cipk3/teb/uvr3/rbb1(Quad mutant)and oePEPC/cipk3/teb/uvr3/rbb1(oePEPC/Quad mutant)were smaller than those of wild type,indicating that knockout of DNA damage sensing and repair-related genes affects Arabidopsis leaf size and petal size.Under normal CO2 and low CO2 conditions,the leaf area and petal length,width and area of oePEPC/cipk3/teb/uvr3/rbb1(oePEPC/Quad mutant)lines were larger than those of cipk3/teb/uvr3/rbb1(Quad mutant),suggesting that transgenic maize C4-PEPC gene might effectively enhance photosynthesis in plants.2.So far,we have obtained the 9th generations(MA-8).The next step is to obtain 11th generations of the Arabidopsis mutant accumulation lines(Col-0?cipk3/teb/uvr3/rbb1?oePEPC/cipk3/teb/uvr3/rbb1),under normal CO2 and low CO2 conditions.We will perform genome resequencing to track genetic change,and provides a genome editing solution for future introduction of C4 into C3 crops.