Research On The Function And Interference Of Salinity Related Genes In Apostichopus Japonicus

Apostichopus japonicas is a kind of valuable marine product in China. With the continuous expansion of the scale of cultured Apostichopus japonicas, cultured Apostichopus japonicas appeared of germplasm resource degradation, and disease resistance decreased significantly, as well as the reduction of output, disease and poor growth caused by water temperature, salinity and other factors. Salinity of pond located near the rivers was easy to be reduced as a result of rainfall, which would be result in large-scale morbidity and mortality of Apostichopus japonicas, and lead to great economic losses to the Apostichopus japonicas aquaculture. In this study, RNAi technology systems of LYZ, NKA and NKCC1 genes in Apostichopus japonicas were constructed to research theirs function in the salinity traits adaptation of Apostichopus japonicas.(1) In this study, RNA interference recombinant plasmids that Rip-LYZ-1, Rip-LYZ-2 and Rip-LYZ-3 to LYZ, Rip-NKA-1、Rip-NKA-2 and Rip-NKA-3 to NKA, as well as Rip-NKCC-1、Rip-NKCC-2 and Rip-NKCC-3 to NKCC1 were constructed. Then, RNA interference recombinant plasmids were transfected into the primary cultured cells of body cavity fluid to determine the silencing efficiency of all these RNA interference recombinant plasmids to target genes. The relative expression and enzyme activity of target genes were determined to detect the best RNA interference recombinant plasmids. These results showed that: the silencing efficiency of ip-LYZ-1, Rip-LYZ-2 and Rip-LYZ-3 to LYZ were 40 %, 45 %, 0 %; the silencing efficiency of Rip-NKA-1、Rip-NKA-2 and Rip-NKA-3 to NKA were 32 %, 46 %, 39 %; the silencing efficiency of Rip-NKCC-1、Rip-NKCC-2 and Rip-NKCC-3 to NKCC1 were 18%, 51 %, 11 %. Otherwise, Rip-LYZ-2 and Rip-NKA-2 produced a very significant(P <0.01) inhibition to the expression of target gene. The result provides an important reference for in vitro and in vivo RNA interference research.(2) In this experiment, one of the best RNA interference recombinant plasmids-Rip-LYZ-2 were used to take the in vivo RNA interference to construct the in vivo RNA interference technology system of Apostichopus japonicas.Transfected Rip-LYZ-2 into Apostichopus japonicas by oral injection and body cavity injection, then, body cavity fluid, muscle and tube feet of Apostichopus japonicas were concentrated to measure expression of target genes 48 hours post-injection. These results showed that: the relative expression of LYZ in body cavity fluid, muscle and tube feet by body cavity injection were 1.3、560 �'� 0.27; and that by oral injection were 0.58、0.24 �'� 1.6. It indicates that transfect the RNA interference recombinant plasmid by oral injection could get RNA interference that have more stable and higher silencing efficiency.Secondly, transfected respectively with 0.5μg、5μg、10μg and 25μg Rip-LYZ-2 by oral injection. Then, body cavity fluid, muscle and tube feet of Apostichopus japonicas were concentrated to measure expression of target genes 48 hours post-injection. These results showed that: the relative expression of LYZ in body cavity fluid, muscle and tube feet injected with 0.5μg Rip-LYZ-2 were 1.38、2.69 and 4.92; the relative expression of LYZ in body cavity fluid, muscle and tube feet injected with 5μg Rip-LYZ-2 were 2.24、0.78 and 4.55; the relative expression of LYZ in body cavity fluid, muscle and tube feet injected with 10 μg Rip-LYZ-2 were 0.08、1.17 and 0.15; and the relative expression of LYZ in body cavity fluid, muscle and tube feet injected with 25 μg Rip-LYZ-2 were 0.6、0.13 and 0.04. It indicates that: it could be have a stable RNA interference when the injected dose of RNA interference recombinant plasmid reaches 10 μg.At last, transfected Rip-LYZ-2 into Apostichopus japonicas by oral injection, then, body cavity fluid, muscle, body wall, parapodum and tube feet of Apostichopus japonicas were concentrated to measure expression of target genes at 0 h、12 h、24 h、48 h、4 d and 8d post-injection. These results showed that: the relative expression of LYZ in body cavity fluid and muscle were decreased in the first 48 hours and then rapid increased, the lysozyme activity in it was steady declined, but that in muscle was increased at first, and didn`t determine it at 24 h and 48 h, and then rapid increased; on the whole that in body wall was first rose and then fell again on an upward trend; the relative expression and lysozyme activity of LYZ in parapodum reached the minimum at 48 h, then recovery; on the whole the expression of LYZ in tube feet was first rose and then fell again on an upward trend, and lysozyme activity of LYZ in it was ready declined after 12 h. It indicates that: the RNA interference in Apostichopus japonicas is systemic, which is local injection of RNA interference recombinant plasmid could cause RNA interference in different tissues and organs of individual animals, and the RNA interference in Apostichopus japonicas could last about four days.(3) The in vivo RNA interferences of LYA, NKA and NKCC1 genes under the condition of low salinity were carried on in this experiment. And the body cavity fluid of Apostichopus japonicas was extracted at 0 h, 12 h, 24 h, 48 h, 72 h, 96 h post-injection to measure the expression change of target genes. These results showed that: the expressiom of LYZ in LYZ test was significant lower than the conrol groups at 24 h and 72h(P<0.05), lysozyme activity in LYZ test was significant lower than the conrol groups at 24 h and 48h(P<0.05), besides, the expressiom of NKA and NKCC1 in LYZ test was significant lower than the conrol groups(P<0.05); the expressiom of NKA in NKA test was significant lower than the conrol groups at 12 h and 24h(P<0.05), Na~+,K~+-ATPase activity in NKA test was significant lower than the conrol groups at 24 h and 96h(P<0.05), besides, the expressiom of NKCC1 in NKA test was significant lower than the conrol groups from 12 h to 72h(P<0.05), the expressiom of LYZ and lysozyme activity in NKA test was significant lower than the conrol groups at part time; the expressions of NKCC1 and NKA were significant respectively lower than the conrol groups in the first 48 h and 24h; othersise, the ability to regulate osmotic pressure of coelomic cell of Apostichopus japonicas in the test groups was declined according to determine the concentration of Na~+、K~+ and Cl- of body cavity fluid in the test and control groups. It has prove the feature of LYZ, NKA and NKCC1 in salinity traits adaptation of Apostichopus japonicas and there is a complicated interaction mechanism between LYZ, NKA and NKCC1.