Research On The Formation Mechanism Of Calcium,Iron And Zinc Nutrition In Leymus Chinensis And The Establishment Of Virus Induced Gene Silencing System

Mineral elements content of herbage is directly related to the health of livestock,and also indirectly influenced the nutrition of human beings.Leymus chinensis is a kind of wild high quality forage widely distributed in the grasslands of northern China.It plays an important role in the development of grassland animal husbandry and grassland restoration.In this study,we investigated the mechanism of calcium,iron and zinc nutrition formation of Leymus chinensis,the effects of exogenous CaCl₂on the photosynthesis and feeding quality of Leymus chinensis,and its regulatory role in the response to abiotic stress.In addition,we established a barley stripe mosaic virus?BSMV?-mediated transient gene overexpression system in Leymus chinensis.This method would provide technical support for the study of functional genomics of Leymus chinensis.The main results are as follows:1.Research on the content of Ca,Fe and Zn in shoots of Leymus chinensis in natural grassland.In a sample plot of Xilingol League,among the 20 investigated plants,the Ca content of Leymus chinensis in shoots ranked 18^th,Fe content ranked3^rdd and Zn content ranked 5^th.In the other plot,among the 11 plants investigated,the Ca content of Leymus chinensis in shoots ranked 10^th,Fe content ranked 8^thh and Zn content ranked 7^th.In addition,the content of Ca,Fe and Zn in shoots among 6populations of Leymus chinensis in Inner Mongolia was significantly different.The results indicated that compared with other associated plants,the content of Ca,Fe and Zn of Leymus chinensis in shoots varied from place to place.2.The effect of mowing frequency,grazing intensity,combined grazing with cattle and sheep and fertilizering on the content of Ca,Fe and Zn in shoots of Leymus chinensis.By mowing of Leymus chinensis with different frequency for 19consecutive years,the results showed that mowing promoted the the accumulation of Ca,but not affected the accumulation of Fe and Zn.Ujimqin sheep were seclected as the grazing livestock,grazing intensity did not affect the accumulation of Ca and Zn in Leymus chinensis,but the accumulation of Fe was promoted under heavy grazing.Grazing with different combination of cattle and sheep did not affect the accumulation of Ca and Zn in shoots of Leymus chinensis,but promoted the accumulation of Fe.Short-time fertilization did not affect the accumulation of Ca,Fe and Zn in shoots of Leymus chinensis.3.The effect of mineral elements in roots on the content of Ca in shoots and roots of Leymus chinensis.As the concentration of Ca²⁺ranged from 0.01 mM to 1mM,the content of Ca in shoots and roots of Leymus chinensis increased 20%⁴0%.As the concentration of Ca²⁺ranged from 1 mM to 20 mM,Ca mainly accumulated in roots.For NO₃^-,the optimum concentration of Ca accumulation in shoots and roots was 5 mM.As the concentration of NH₄⁺ranged from 1 mM to 20 mM or PO₄^3-ranged from 0.01 mM to 1 mM,it did not affect the accumulation of Ca in shoots and roots.20 mM PO₄^3-significantly inhibited the accumulation of Ca in shoots and roots.As the concentration of K⁺ranged from 0.01 mM to 5 mM,it did not affect the accumulation of Ca in shoots and roots.20 mM K⁺significantly inhibited the accumulation of Ca in shoots.1¹0 mM Mg²⁺could inhibit the accumulation of Ca in shoots and roots.For Fe²⁺,the optimum concentration of Ca accumulation in shoots and roots was 0.05 mM.0.5¹ mM Fe²⁺could inhibit the accumulation of Ca in shoots.Zn²⁺ranged from 1.5?M to 75?M did not affect the accumulation of Ca in shoots and roots.0.15?M Zn²⁺could inhibit the accumulation of Ca in shoots.4.The effect of mineral elements in roots on the content of Fe in shoots and roots of Leymus chinensis.As the concentration of Fe²⁺rised from 0.0005 mM to 1mM,it significantly promoted the accumulation of Fe in shoots and roots,and with clear concentration dependence.For NO₃^-,the optimum concentration of Fe accumulation in shoots was 5 mM.As the concentration of NO₃^-ranged from 0.01mM to 20 mM,it did not affect the accumulation of Fe in roots.As the concentration of NH₄⁺ranged from 1 mM to 20 mM,it could promote the accumulation of Fe in shoots,but did not affect the accumulation of Fe in roots.20 mM PO₄^3-significantly inhibited the accumulation of Fe in shoots and roots.For K⁺,the optimum concentration of Fe accumulation in shoots was from 1 mM to 5 mM.As the concentration of K⁺ranged from 0.01 mM to 20 mM,it did not affect the accumulation of Fe in roots.As the concentration of Ca²⁺ranged from 0.01 mM to 20mM,it did not affect the accumulation of Fe in shoots and roots.For Mg²⁺,the optimum concentration of Fe accumulation in roots was 0.01 mM.10 mM Mg²⁺could inhibit the accumulation of Fe in shoots and roots.For Zn²⁺,the optimum concentration of Fe accumulation in shoots was 15?M.As the concentration of Zn²⁺ranged from 0.15?M to 75?M,it did not affect the accumulation of Fe in roots.5.The effect of mineral elements in roots on the content of Zn in shoots and roots of Leymus chinensis.In general,the content of NO₃^-,PO₄^3-,K⁺,and Ca²⁺in the nutrient solution did not affect the accumulation of Zn in shoots and roots.As the concentration of Zn²⁺rised from 0.15?M to 75?M,Zn mainly accumulated in roots.As the concentration of NH₄⁺ranged from 1 mM to 20 mM,it could inhibit the accumulation of Zn in shoots and roots.For Mg²⁺,the optimum concentration of Zn accumulation in roots was 0.01 mM.10 mM Mg²⁺could inhibit the accumulation of Zn in shoots and roots.For Fe²⁺,the optimum concentration of Zn accumulation in shoots and roots ranged from 0.5?M to 50?M.As the concentration of Fe²⁺ranged from 0.5 mM to 1 mM,it could inhibit the accumulation of Zn in shoots and roots.6.Regulation of Ca²⁺on photosynthetic efficiency,feeding quality and abiotic resistance of Leymus chinensis.As the concentration of Ca²⁺in nutrient solution ranged from 0 mM to 20 mM,it had no effect on the main parameters of photosynthetic efficiency,the content of crude protein,crude fat,crude fiber,neutral detergent fiber and acid detergent fiber.But the content of acid detergent lignin in shoots was negatively correlated with the concentration of Ca²⁺.Increasing the content of Ca²⁺in the medium could improve the ability of salt resistance and cold resistance of Leymus chinensis,but the resistance to alkali and osmotic stress of Leymus chinensis was not improved.7.Establishment of barley stripe mosaic virus mediated transient gene overexpression system in Leymus chinensis.In this study,we successfully obtained the four-component vector with ligation independent cloning?LIC?by modifying the three-component vector of gene silencing vector induced by Barley stripe mosaic virus.We chose mCherry,GFP and other fluorescent protein genes as marker genes.Agrobacterium tumefaciens-mediated transient transformation by infiltration was used to transform N.benthamiana.Results showed that the four-component BSMV vector could express two foreign fluorescent protein genes at the same time and last for more than 7 days.After harvested and grinded the spot-agroinfiltrated leaves,they were used to infect the seedlings of Leymus chinensis.The results showed that the foreign fluorescent protein genes could also be expressed in Leymus chinensis.This indicated that BSMV mediated transient gene overexpression system was successfully established in Leymus chinensis.